Collective effects of rising average temperatures and heat events on oviparous embryos.

Survival of the immobile embryo in response to rising temperature is important to determine a species' vulnerability to climate change. However, the collective effects of 2 key thermal characteristics associated with climate change (i.e., rising average temperature and acute heat events) on embryonic survival remain largely unexplored. We used empirical measurements and niche modeling to investigate how chronic and acute heat stress independently and collectively influence the embryonic survival of lizards across latitudes. We collected and bred lizards from 5 latitudes and incubated their eggs across a range of temperatures to quantify population-specific responses to chronic and acute heat stress. Using an embryonic development model parameterized with measured embryonic heat tolerances, we further identified a collective impact of embryonic chronic and acute heat tolerances on embryonic survival. We also incorporated embryonic chronic and acute heat tolerance in hybrid species distribution models to determine species' range shifts under climate change. Embryos' tolerance of chronic heat (T-chronic) remained consistent across latitudes, whereas their tolerance of acute heat (T-acute) was higher at high latitudes than at low latitudes. Tolerance of acute heat exerted a more pronounced influence than tolerance of chronic heat. In species distribution models, climate change led to the most significant habitat loss for each population and species in its low-latitude distribution. Consequently, habitat for populations across all latitudes will shift toward high latitudes. Our study also highlights the importance of considering embryonic survival under chronic and acute heat stresses to predict species' vulnerability to climate change.

Efectos colectivos del aumento de las temperaturas promedio y los eventos de calor en embriones ovíparos Resumen La supervivencia de los embriones inmóviles en respuesta al incremento de temperatura es importante para determinar la vulnerabilidad de las especies al cambio climático. Sin embargo, los efectos colectivos de dos características térmicas claves asociadas con el cambio climático (i. e., aumento de temperatura promedio y eventos de calor agudo) sobre la supervivencia embrionaria permanecen en gran parte inexplorados. Utilizamos mediciones empíricas y modelos de nicho para investigar cómo el estrés térmico crónico y agudo influye de forma independiente y colectiva en la supervivencia embrionaria de los lagartos en todas las latitudes. Recolectamos y criamos lagartos de cinco latitudes e incubamos sus huevos en un rango de temperaturas para cuantificar las respuestas específicas de la población al estrés por calor crónico y agudo. Posteriormente, mediante un modelo de desarrollo embrionario parametrizado con mediciones de tolerancia embrionaria al calor, identificamos un impacto colectivo de las tolerancias embrionarias al calor agudo y crónico en la supervivencia embrionaria. También incorporamos la tolerancia embrionaria crónica y aguda al calor en modelos de distribución de especies híbridas para determinar los cambios de distribución de las especies bajo el cambio climático. La tolerancia embrionaria al calor crónico (T­crónico) permaneció constante, mientras que la tolerancia al calor agudo (T­agudo) fue mayor en latitudes altas que en latitudes bajas. La tolerancia al calor agudo ejerció una influencia más pronunciada que la tolerancia al calor crónico. En los modelos de distribución de especies, el cambio climático provocó la pérdida de hábitat más significativa para cada población y especie en su distribución de latitudes bajas. En consecuencia, el hábitat para poblaciones en todas las latitudes se desplazará a latitudes altas. Nuestro estudio también resalta la importancia de considerar la supervivencia embrionaria bajo estrés térmico crónico y agudo para predecir la vulnerabilidad de las especies al cambio climático.

A Novel Electrochemical Sensor Based on Pd Confined Mesoporous Carbon Hollow Nanospheres for the Sensitive Detection of Ascorbic Acid, Dopamine, and Uric Acid.

In this study, we designed a novel electrochemical sensor by modifying a glass carbon electrode (GCE) with Pd confined mesoporous carbon hollow nanospheres (Pd/MCHS) for the simultaneous detection of ascorbic acid (AA), dopamine (DA), and uric acid (UA). The structure and morphological characteristics of the Pd/MCHS nanocomposite and the Pd/MCHS/GCE sensor are comprehensively examined using SEM, TEM, XRD and EDX. The electrochemical properties of the prepared sensor are investigated through CV and DPV, which reveal three resolved oxidation peaks for AA, DA, and UA, thereby verifying the simultaneous detection of the three analytes. Benefiting from its tailorable properties, the Pd/MCHS nanocomposite provides a large surface area, rapid electron transfer ability, good catalytic activity, and high conductivity with good electrochemical behavior for the determination of AA, DA, and UA. Under optimized conditions, the Pd/MCHS/GCE sensor exhibited a linear response in the concentration ranges of 300-9000, 2-50, and 20-500 µM for AA, DA, and UA, respectively. The corresponding limit of detection (LOD) values were determined to be 51.03, 0.14, and 4.96 µM, respectively. Moreover, the Pd/MCHS/GCE sensor demonstrated outstanding selectivity, reproducibility, and stability. The recovery percentages of AA, DA, and UA in real samples, including a vitamin C tablet, DA injection, and human urine, range from 99.8-110.9%, 99.04-100.45%, and 98.80-100.49%, respectively. Overall, the proposed sensor can serve as a useful reference for the construction of a high-performance electrochemical sensing platform.

Heterospecific eavesdropping on disturbance cues of a treefrog.

Alarm signals and cues are crucial to animal survival and vary greatly across species. Eavesdropping on heterospecific alarm signals and cues can provide eavesdroppers with information about potential threats. In addition to acoustic alarm signals, evidence has accumulated that chemical alarm cues and disturbance cues can also play a role in alerting conspecifics to potential danger in adult anurans (frogs and toads). However, there is very little known about whether disturbance cues are exploited by heterospecifics. In the present study, we conducted a binary choice experiment and a prey chemical discrimination experiment, respectively, to test the responses of a sympatric anuran species (red webbed treefrogs, Rhacophorus rhodopus) and a sympatric predator species (Chinese green tree vipers, Trimeresurus stejnegeri) to disturbance odors emitted by serrate-legged small treefrogs (Kurixalus odontotarsus). In the binary choice experiment, we found that the presence of disturbance odors did not significantly trigger the avoidance behavior of R. rhodopus. In the prey chemical discrimination experiment, compared with odors from undisturbed K. odontotarsus (control odors) and odorless control, T. stejnegeri showed a significantly higher tongue-flick rate in response to disturbance odors. This result implies that disturbance odor cues of K. odontotarsus can be exploited by eavesdropping predators to detect prey. Our study provides partial evidence for heterospecific eavesdropping on disturbance cues and has an important implication for understanding heterospecific eavesdropping on chemical cues of adult anurans.

Keep numbers in view: red-eared sliders (Trachemys scripta elegans) learn to discriminate relative quantities.

The ability to discriminate relative quantities, one of the numerical competences, is considered an adaptive trait in uncertain environments. Besides humans, previous studies have reported this capacity in several non-human primates and birds. Here, we test whether red-eared sliders (Trachemys scripta elegans) can discriminate different relative quantities. Subjects were first trained to distinguish different stimuli with food reward. Then, they were tested with novel stimulus pairs to demonstrate how they distinguished the stimuli. The results show that most subjects can complete the initial training and use relative quantity rather than absolute quantity to make choices during the testing phase. This study provides behavioural evidence of relative quantity discrimination in a reptile species and suggests that such capacity may be widespread among vertebrates.

A fast and robust iterative genome-editing method based on a Rock-Paper-Scissors strategy.

The production of optimized strains of a specific phenotype requires the construction and testing of a large number of genome modifications and combinations thereof. Most bacterial iterative genome-editing methods include essential steps to eliminate selection markers, or to cure plasmids. Additionally, the presence of escapers leads to time-consuming separate single clone picking and subsequent cultivation steps. Herein, we report a genome-editing method based on a Rock-Paper-Scissors (RPS) strategy. Each of three constructed sgRNA plasmids can cure, or be cured by, the other two plasmids in the system; plasmids from a previous round of editing can be cured while the current round of editing takes place. Due to the enhanced curing efficiency and embedded double check mechanism, separate steps for plasmid curing or confirmation are not necessary, and only two times of cultivation are needed per genome-editing round. This method was successfully demonstrated in Escherichia coli and Klebsiella pneumoniae with both gene deletions and replacements. To the best of our knowledge, this is the fastest and most robust iterative genome-editing method, with the least times of cultivation decreasing the possibilities of spontaneous genome mutations.

The Characterization of Mitochondrial Genome of Spotted Pond Turtle (Geoclemys hamiltonii).

The spotted pond turtle Geoclemys hamiltonii (Gray, 1830) is widely distributed in the Indus, Ganges, and Brahmaputra river basins. In this study, the complete mitochondrial genome (mitogenome) of G. hamiltonii was sequenced using the next-generation sequencing (NGS) and Sanger sequencing, and the essential characteristics, gene arrangement, and phylogenetic relationship were analyzed. The results showed that the G. hamiltonii mitogenome was 16,505 bp in length (A: 33.6%, C: 27.1%, G: 13.4%, T: 25.8%) and consisted of 22 tRNAs, 13 protein-coding genes, two ribosomal RNA genes, and a non-coding control region (GenBank accession ON243873). The genome composition of G. hamiltonii presented a slight A + T bias (59.4%), and showed a positive AT skew (0.131) and a negative GC skew (- 0.338). All tRNAs had the typical clover structure, except trnS1 (GCT). The gene order of the G. hamiltonii mitogenome was the same as other Geoemydidae mitogenomes. A phylogenetic analysis based on the complete mitogenome indicated that the G. hamiltonii grouped independently of other species in the family Geoemydidae, supporting the species' placement in the monotypic genus Geoclemys. Our results describe a novel genome at the species level. As the first complete mitogenome of G. hamiltonii, it provided valuable molecular information for phylogenetic and conservation genetics analyses of G. hamiltonii.

Crystallization, X-ray diffraction analysis and structure of ICMP from Pseudomonasaeruginosa.

Insulin-cleaving membrane protease (ICMP), an outmember protein of Pseudomonas aeruginosa (P. aeruginosa), plays a critical role in the pathogenesis of the bacterium. ICMP has been reported to be involved in the process of iron uptake. In this study, we report the high-resolution structure of ICMP determined by single-wavelength anomalous diffraction (SAD), which shows an atypical HxxE motif that differs from the canonical zinc dependent M75 peptidases and a "V-shaped" cleft that is observed to coordinate the metal ion for the first time. Crystals from the selenomethionine-substituted ICMP(Se-Met ICMP) diffract to 1.9 Å resolution and belong to space group P21, with unit-cell parameters a = 87.93, b = 78.14, c = 9.92 Å, α = 90°, ß = 113.5°, γ = 90°. ICMP consists of two up-and-down helix bundles, which are arranged into an inverted "V" shape. Unexpectedly, no electron densities of metal ions are observed around the ICMP HxxE motif, which is shown to be involved in metal coordination in zinc-dependent M75 peptidases. In contrast, we find a metal ion at the opening cleft of the V-shaped structure of ICMP, where the ICMP residues Asp211, Glu316, Cys319, Asp322, and Asp397 are observed to coordinate the metal via hydrogen-bond interactions. Such observations might imply new potential substrate-binding and catalytic sites. The current work therefore provides novel insights into the diversity of the HxxE-motif-containing peptidase and paves the way for future studies aiming to delineate the mechanism of ICMP catalysis.

Association between air pollution and hospital admissions for chronic respiratory disease in people aged over 65 years: a time series analysis in Ningbo, China, 2015-2017.

PURPOSE: To provide essential information of environmental triggers leading to CRD. METHODS: We investigated the short-term effects of ambient air pollutants on CRD-related hospitalizations in people aged ≥ 65 years in Ningbo. Data on 23,610 cases of CRD requiring hospitalization were collected from January 2015 to August 2017. After adjusting for temporal trends, seasonality, meteorological conditions, day of week (DOW), and public holidays, we used generalized additive Poisson distribution models to calculate the excess risks (ERs) and 95% confidence intervals (95% CIs) of CRD related hospitalizations. RESULTS: Our results showed that fine particulate matter (PM2.5), inhalable particulate matter (PM10), sulfur dioxide (SO2), nitrogen dioxide (NO2), and carbon monoxide (CO) were associated with CRD-related hospitalizations in people aged ≥ 65 years. We observed that each 10 µg/m3 increase (except for each 0.1 mg/m3 increase in CO) in the concentration of air pollutants, the percentage of CRD-related hospitalizations due to PM2.5, PM10, and SO2 exposure at lag 07, NO2 exposure at lag 03, and CO exposure at lag 0 increased by 2.13% (95% CI: 0.55%, 3.74%), 1.76% (95% CI: 0.70%, 2.83%), 8.24% (95% CI: 0.92%, 16.09%), 2.16% (95% CI: 0.26%, 4.05%), and 1.19% (95% CI: 0.26%, 2.12%), respectively. In addition, we found stronger effects of particulate matter in 75-84 years age group, on warmer days, and in asthmatics. CONCLUSION: In conclusion, air pollution may have adverse effects on CRD-related hospitalizations among people aged ≥ 65 years in Ningbo. Therefore, public health measures should be taken to improve air quality.

The Genome of Shaw's Sea Snake (Hydrophis curtus) Reveals Secondary Adaptation to Its Marine Environment.

The transition of terrestrial snakes to marine life ∼10 Ma is ideal for exploring adaptive evolution. Sea snakes possess phenotype specializations including laterally compressed bodies, paddle-shaped tails, valvular nostrils, cutaneous respiration, elongated lungs, and salt glands, yet, knowledge on the genetic underpinnings of the transition remains limited. Herein, we report the first genome of Shaw's sea snake (Hydrophis curtus) and use it to investigate sea snake secondary marine adaptation. A hybrid assembly strategy obtains a high-quality genome. Gene family analyses date a pulsed coding-gene expansion to ∼20 Ma, and these genes associate strongly with adaptations to marine environments. Analyses of selection pressure and convergent evolution discover the rapid evolution of protein-coding genes, and some convergent features. Additionally, 108 conserved noncoding elements appear to have evolved quickly, and these may underpin the phenotypic changes. Transposon elements may contribute to adaptive specializations by inserting into genomic regions around functionally related coding genes. The integration of genomic and transcriptomic analyses indicates independent origins and different components in sea snake and terrestrial snake venom; the venom gland of the sea snake harbors the highest PLA2 (17.23%) expression in selected elapids and these genes may organize tandemly in the genome. These analyses provide insights into the genetic mechanisms that underlay the secondary adaptation to marine and venom production of this sea snake.

De novo sequence redesign of a functional Ras-binding domain globally inverted the surface charge distribution and led to extreme thermostability.

To acquire extremely thermostable proteins of given functions is challenging for conventional protein engineering. Here we applied ABACUS, a statistical energy function we developed for de novo amino acid sequence design, to globally redesign a Ras-binding domain (RBD), and obtained an extremely thermostable RBD that unfolds reversibly at above 110°C, the redesigned RBD experimentally confirmed to have expected structure and Ras-binding interface. Directed evolution of the redesigned RBD improved its Ras-binding affinity to the native protein level without excessive loss of thermostability. The designed amino acid substitutions were mostly at the protein surface. For many substitutions, strong epistasis or significantly differentiated effects on thermostability in the native sequence context relative to the redesigned sequence context were observed, suggesting the globally redesigned sequence to be unreachable through combining beneficial mutations of the native sequence. Further analyses revealed that by replacing 38 of a total of 48 non-interfacial surface residues at once, ABACUS redesign was able to globally "invert" the protein's charge distribution pattern in an optimized way. Our study demonstrates that computational protein design provides powerful new tools to solve challenging protein engineering problems.

Noise constrains the evolution of call frequency contours in flowing water frogs: a comparative analysis in two clades.

BACKGROUND: The acoustic adaptation hypothesis (AAH) states that signals should evolve towards an optimal transmission of the intended information from senders to intended receivers given the environmental constraints of the medium that they traverse. To date, most AAH studies have focused on the effect of stratified vegetation on signal propagation. These studies, based on the AAH, predict that acoustic signals should experience less attenuation and degradation where habitats are less acoustically complex. Here, we explored this effect by including an environmental noise dimension to test some AAH predictions in two clades of widespread amphibians (Bufonidae and Ranidae) that actively use acoustic signals for communication. By using data from 106 species in these clades, we focused on the characterization of the differences in dominant frequency (DF) and frequency contour (i.e., frequency modulation [FM] and harmonic performances) of mating calls and compared them between species that inhabit flowing-water or still-water environments. RESULTS: After including temperature, body size, habitat type and phylogenetic relationships, we found that DF differences among species were explained mostly by body size and habitat structure. We also showed that species living in lentic habitats tend to have advertisement calls characterized by well-defined FM and harmonics. Likewise, our results suggest that flowing-water habitats can constrain the evolutionary trajectories of the frequency-contour traits of advertisement calls in these anurans. CONCLUSIONS: Our results may support AAH predictions in frogs that vocalize in noisy habitats because flowing-water environments often produce persistent ambient noise. For instance, these anurans tend to generate vocalizations with less well-defined FM and harmonic traits. These findings may help us understand how noise in the environment can influence natural selection as it shapes acoustic signals in affected species.

Conspecific odor cues induce different vocal responses in serrate-legged small treefrogs, but only in the absence of acoustic signals.

BACKGROUND: Signal detection is crucial to survival and successful reproduction, and animals often modify behavioral decisions based on information they obtained from the social context. Undeniably, the decision-making in male-male competition and female choice of anurans (frogs and toads) depends heavily on acoustic signals. However, increasing empirical evidence suggests that additional or alternative types of cue (e.g., visual, chemical, and vibratory) can be used to detect, discriminate and locate conspecifics in many anuran species. Nevertheless, few studies have investigated whether conspecific odor cues affect male's calling behavior. In this study, we conducted an experiment to investigate whether and how different chemical cues (male odors, female odors, and stress odors) from conspecifics affect male's calling strategies in serrate-legged small treefrogs (Kurixalus odontotarsus), and whether the combined chemical and acoustic stimuli have additive effects on calling behavior or not. RESULTS: We found that compared with female odors, male K. odontotarsus reduced calling investment in response to male odors or stress odors, in the absence of rival's advertisement calls. When odor stimuli and advertisement calls were presented simultaneously, however, there were no differences in the vocal response of focal males among odor groups. CONCLUSIONS: These results provide evidence that male treefrogs switch calling investment according to different odor cues from conspecifics, and further demonstrate that calling behavior can be affected by chemical cues in anuran species. Our study highlights the potential role of airborne chemical cues in sex identification and contributes to increase our understanding of anuran communication.

Multisensory modalities increase working memory for mating signals in a treefrog.

Animal choruses, such as those found in insects and frogs, are often intermittent. Thus, females sampling males in the chorus might have to remember the location of the potential mates' calls during periods of silence. Although a number of studies have shown that frogs use and prefer multimodal mating signals, usually acoustic plus visual, it is not clear why they do so. Here we tested the hypothesis that preference for multimodal signals over unimodal signals might be due to multimodal signals instantiating longer memories than unimodal signals, particularly during the inter-chorus intervals. We tested this hypothesis in serrate-legged small treefrogs Kurixalus odontotarsus whose males produce advertisement calls accompanied by conspicuous vocal sac inflation. Females were tested with acoustic and acoustic + visual (video of inflating-deflating vocal sac) mating calls. We found that females prefer multimodal calls over unimodal, audio-only calls. Furthermore, multimodal calls are still preferred after a silent period of up to 30 s, a time that spans the average silent period of the chorus. This was not true of unimodal calls. Our results demonstrate that a multimodal signal can engage longer working memory than a unimodal signal, and thus female memory might favour the evolution of multimodal signals in males through sexual selection. Selection might also favour female preference for multimodal signals if longer memory facilitates mate searching and assessment. Our study does not allow us to elucidate the sequence of evolution of this trait and preference.

pH-controlled mechanism of photocatalytic RhB degradation over g-C3N4 under sunlight irradiation.

Photocatalysis of dye degradation is one of green and cheap technologies for solving environmental pollution. Whereas it is rarely concerned that the degradation process varied with the change of solution condition, this work studied the influence of hydrion in the solution on the photodegradation process of Rhodamine B (RhB) over g-C3N4. The photocatalytic activity of RhB degradation was enhanced gradually with increased hydrion content in the system. The efficiency for RhB degradation over g-C3N4 in weak acidic system with interference of multiple metal-ions still reached near 95% after 30 min of natural sunlight irradiation. A large amount of oxidation species and the hydroxylation mineralization process were induced by increasing the hydrion concentration. Two degradation processes for deethylation of four ethyl groups and the direct chromophoric degradation were discovered and proved by multifarious intermediates in different systems using the ESR technique, LC/MS and GC/MS analysis. In addition, the photosensitization played a critical role in the RhB degradation. A feasible degradation mechanism was proposed for the RhB degradation based on the experimental results.

A simple strategy for the detection of Pb(II) and Cu(II) by an electrochemical sensor based on Zn/Ni-ZIF-8/XC-72/Nafion hybrid materials.

In this study, a novel electrochemical sensor for simultaneous detection of Pb(II) and Cu(II) was constructed by using Zn/Ni-ZIF-8/XC-72/Nafion hybrid material as electrode surface modifier. XRD, FT-IR, XPS and SEM were used to study the crystal structure, functional groups, element types and morphologies of the prepared materials. The electrochemical performance of the Zn/Ni-ZIF-8/XC-72/Nafion/GCE sensor were investigated by CV, EIS and DPV. In addition, the effects of various conditions including pH, the type of buffer and the ratio of Zn/Ni-ZIF-8 to XC-72 were also explored for the determination of Pb(II) and Cu(II). Under the optimum conditions, the constructed sensor exhibited outstanding linear response of Pb(II) (0.794-39.6 ppm) and Cu(II) (0.397-19.9 ppm) with detection limits of 0.0150 and 0.0096 ppm, respectively. Finally, the fabricated sensor was further used to detect Pb(II) and Cu(II) in real samples, and the satisfactory recovery was obtained.

Microbial community and molecular ecological network in the EGSB reactor treating antibiotic wastewater: Response to environmental factors.

In this study, one lab-scale EGSB reactor (1.47 L volume) was designed to treat the antibiotic wastewater under different environmental factors, including the addition of cephalexin (CFX), Temperature (T) and Hydraulic Retention Time (HRT). The microbial community structure in EGSB reactor was analyzed with high-throughput sequencing technology to investigate their response to environmental factors changes, and then the random-matrix-theory (RMT)-based network analysis was used to investigate the microbial community's molecular ecological network in EGSB systems treating antibiotics wastewater. Moreover, the explanatory value of each environmental factor on the change of microbial community structure was obtained through the result of redundancy analysis (RDA). The results showed that the addition of cephalexin (CFX), decline of T and decline of HRT (8 h) would decrease the removal efficiency of COD decreasing. And the removal efficiency of CFX would not be affected by decline of T and HRT, except the producing and degrading process of CFX by-products was changed obviously. The result of RDA analysis suggested the environmental factors mainly affected bacterial and fungal microbial community structure but not archaeal ones. The result of high-throughput sequencing showed the relative abundance (RA) of Firmicutes had been obviously affected by T and HRT, which might be main reason leading to the decrease of COD removal efficiency. In addition, molecular ecological network analysis showed the growth of Bacteroidetes occupied the niche of functional microorganism and led to the unstable operation of EGSB when T declined. What's more, the molecular ecological network analysis revealed that Exophiala which belonged to fungi Ascomycota phylum was the hub genus to degrade complex refractory organic pollutants, and Aceticlastic methanogens Methanosaeta was the core functional archaea genus.

Ontogenetic development of hearing sensitivity to airborne sound in the female red-eared slider, Trachemys scripta elegans.

Ontogenetic development of hearing sensitivity has been verified in many groups of vertebrates, but not turtles. Turtles exhibit sexual dimorphism in hearing. To examine the development of hearing in female turtles, auditory brainstem responses (ABR) were compared by assessing the hearing-sensitivity bandwidth, ABR threshold, and latency of female Trachemys scripta elegans aged 1 week, 1 month, 1 yr, and 5 yr. The hearing-sensitivity bandwidths were 0.2-1.1, 0.2-1.1, 0.2-1.3, and 0.2-1.4 kHz in each age group, respectively. Below 0.6 kHz, the ABR threshold decreased from the 1-week to 1-yr age group, with a significant difference between age groups. No significant difference was detected between the 1- and 5-yr age groups (within a stimulus frequency of 0.2-0.6 kHz). Above 0.6 kHz, ABR thresholds decreased significantly from the 1-yr to 5-yr age group (within a stimulus frequency of 0.7-1.0 kHz). There was no significant difference between the 1-month and 1-yr age groups (within a stimulus frequency of 0.7-1.0 kHz), or between the 1-week and 1-month age groups (within a stimulus frequency of 0.7-1.0 kHz, except 0.9 kHz). Thus, female turtle hearing shows frequency-segmented development.

Characterization and directed evolution of propionyl-CoA carboxylase and its application in succinate biosynthetic pathway with two CO2 fixation reactions.

Propionyl-CoA carboxylase (PCC) is a promising enzyme in the fields of biological CO2 utilization, synthesis of natrual products, and so on. The activity and substrate specificity of PCC are dependent on its key subunit carboxyltransferase (CT). To obtain PCC with high enzyme activity, seven pccB genes encoding CT subunit from diverse microorganisms were expressed in recombinant E. coli, and PccB from Bacillus subtilis showed the highest activity in vitro. To further optimize this protein using directed evolution, a genetic screening system based on oxaloacetate availability was designed to enrich the active variants of PccBBs. Four amino acid substitutions (D46G, L97Q, N220I and I391T) proved of great assistance in PccBBs activity improvement, and a double mutant of PccBBs (N220I/I391T) showed a 94-fold increase of overall catalytic efficiency indicated by kcat/Km. Moreover, this PccBBs double mutant was applied in construction of new succinate biosynthetic pathway. This new pathway produces succinate from acetyl-CoA with fixation of two CO2 molecules, which was confirmed by isotope labeling experiment with NaH13CO3. Compared with previous succinate production based on carboxylation of phosphoenolpyruvate or pyruvate, this new pathway showed some advantages including higher CO2 fixation potentiality and availability under aerobic conditions. In summary, this study developed a PCC with high enzyme activity which can be widely used in biotechnology field, and also demonstrated the feasibility of new succinate biosynthetic pathway with two CO2 fixation reactions.

Study on Self-Assembled Morphology and Structure Regulation of α-Zein in Ethanol-Water Mixtures.

α-Zein has received widespread attention owing to its unique solubility, amphipathic, and self-assembly properties, which is because of its high proportion of nonpolar amino acids and unique amino acid sequence. The protein self-assembly is a significant and widely observed phenomenon in many scientific areas such as food and biomedicine, among many industries. In this study, we investigated the self-assembly behavior of α-zein and regulated the morphology and structure of the self-assembled α-zein by varying the experimental parameters like pH, ethanol content, induction time, and α-zein concentration during the self-assembly process in ethanol-water mixtures. The nanospheres and nanofibers were observed under different conditions [nanospheres observed under acidic and strongly alkaline (pH > 10.5) conditions or for ethanol content lower than 65% and higher than 75%; nanofibers observed under weakly alkaline (pH 9.5-10.5) conditions or for 65-75% ethanol concentration for induction duration longer than 24 h]. The morphological and structural analyses of the self-assembled α-zein showed that the self-assembly process was accompanied by the transformation of the morphology and conformation of α-zein. The studies on the self-assembly process and mechanism revealed that α-zein first self-assembled into nanospheres, followed by the nanospheres adhering to shape-beaded fibers and finally fibers, accompanied by a structural transformation from the disordered into ordered state. The nanosphere formation is noted to follow the nucleation-based polymerization, and the nanosphere-mediated mechanisms lead to the formation of nanofibers. Moreover, the hydrophobic interactions, hydrogen bonds, and electrostatic interactions are concluded to drive the α-zein self-assembly. The findings from this study are expected to provide a theoretical basis for expanding the commercial applications of α-zein.

Male serrate-legged treefrogs adjust competition strategies according to visual or chemical cues from females.

There is increasing evidence that many anurans use multimodal cues to detect, discriminate and/or locate conspecifics and thus modify their behaviors. To date, however, most studies have focused on the roles of multimodal cues in female choice or male-male interactions. In the present study, we conducted an experiment to investigate whether male serrate-legged small treefrogs (Kurixalus odontotarsus) used visual or chemical cues to detect females and thus altered their competition strategies in different calling contexts. Three acoustic stimuli (advertisement calls, aggressive calls and compound calls) were broadcast in a randomized order after a spontaneous period to focal males in one of four treatment groups: combined visual and chemical cues of a female, only chemical cues, only visual cues and a control (with no females). We recorded the vocal responses of the focal males during each 3 min period. Our results demonstrate that males reduce the total number of calls in response to the presence of females, regardless of how they perceived the females. In response to advertisement calls and compound calls, males that perceived females through chemical cues produced relatively fewer advertisement calls but more aggressive calls. In addition, they produced relatively more aggressive calls during the playback of aggressive calls. Taken together, our study suggests that male Kodontotarsus adjust their competition strategies according to the visual or chemical cues of potential mates and highlights the important role of multisensory cues in male frogs' perception of females.