PARP1 modulates METTL3 promoter chromatin accessibility and associated LPAR5 RNA m6A methylation to control cancer cell radiosensitivity.

Chromatin remodeling and N6-methyladenosine (m6A) modification are two critical layers in controlling gene expression and DNA damage signaling in most eukaryotic bioprocesses. Here, we report that poly(ADP-ribose) polymerase 1 (PARP1) controls the chromatin accessibility of METTL3 to regulate its transcription and subsequent m6A methylation of poly(A)+ RNA in response to DNA damage induced by radiation. The transcription factors nuclear factor I-C (NFIC) and TATA binding protein (TBP) are dependent on PARP1 to access the METTL3 promoter to activate METTL3 transcription. Upon irradiation or PARP1 inhibitor treatment, PARP1 disassociated from METTL3 promoter chromatin, which resulted in attenuated accessibility of NFIC and TBP and, consequently, suppressed METTL3 expression and RNA m6A methylation. Lysophosphatidic Acid Receptor 5 (LPAR5) mRNA was identified as a target of METTL3, and m6A methylation was located at A1881. The level of m6A methylation of LPAR5 significantly decreased, along with METTL3 depression, in cells after irradiation or PARP1 inhibition. Mutation of the LPAR5 A1881 locus in its 3' UTR results in loss of m6A methylation and, consequently, decreased stability of LPAR5 mRNA. METTL3-targeted small-molecule inhibitors depress murine xenograft tumor growth and exhibit a synergistic effect with radiotherapy in vivo. These findings advance our comprehensive understanding of PARP-related biological roles, which may have implications for developing valuable therapeutic strategies for PARP1 inhibitors in oncology.

A pig model of symptomatic spinal epidural hematoma.

PURPOSE: The purpose of this study was to establish an animal model capable of simulating the development and decompression process of symptomatic spinal epidural hematoma (SSEH). METHODS: A total of 16 male Bama miniature pigs were included in this study and randomly allocated into four groups: Group A (4 h 20 mmHg hematoma compression), Group B (4 h 24 mmHg hematoma compression), Group C (4 h 28 mmHg hematoma compression), and Group Sham (control). Real-time intra-wound hematoma compression values were obtained using the principle of connectors. Electrophysiological analyses, including the latency and amplitude of somatosensory evoked potentials (SSEP) and motor evoked potentials (MEP), along with behavioral observations (Tarlov score), were performed to assess this model. RESULTS: ANOVA tests demonstrated significant differences in the latency and relative amplitude of SSEP and MEP between Groups C and Sham after 4 h of hematoma compression and one month after surgery (P < 0.01). Behavioral assessments 8 h after surgery indicated that animals subjected to 28 mmHg hematoma compression suffered the most severe spinal cord injury. Pearson correlation coefficient test suggested a negative correlation between the epidural pressure and Tarlov score (r = -0.700, p < 0.001). With the progression of compression and the escalation of epidural pressure, the latency of SSEP and MEP gradually increased, while the relative amplitude gradually decreased. CONCLUSIONS: When the epidural pressure reaches approximately 24 mmHg, the spinal cord function occurs progressive dysfunction. Monitoring epidural pressure would be an effective approach to assist to identify the occurrence of postoperative SSEH.

Perioperative complications of en bloc resection and anterior column reconstruction for thoracic and lumbar spinal tumors.

PURPOSE: To evaluate the perioperative clinical outcomes of en bloc resection and anterior column reconstruction for thoracolumbar spinal tumors. METHODS: This study conducted a retrospective analysis of prospective data collection of 86 consecutive patients, including 40 males and 46 females, with an average age of 39 years (ranged from 10 to 71 years). There were 35 cases of a malignant primary tumor,42 cases of an aggressive benign tumor, and nine cases of metastases. The main lesions were located in 65 cases of thoracic spine, 17 cases of lumbar spine, and 4 cases of thoracolumbar spine. Tumors involved one level in 45 patients, two levels in 12 patients, three levels in 21 patients, four levels in five patients, five levels in two patients, and six levels in one patient. RESULTS: According to the Weinstein-Boriani-Biagini surgical staging system, all patients achieved en bloc resections, including 74 cases of total en bloc spondylectomy and 12 cases of sagittal resections. The mean surgical time was 559 min (210-1208 min), and the mean total blood loss was 1528 ml (260-5500 ml). A total of 122 complications were observed in 62(72.1%) patients, of which 18(20.9%) patients had 25 major complications and one patient (1.2%) died of complications. The combined approach (P = 0.002), total blood loss (P = 0.003), staged surgery (P = 0.004), previous surgical history (P = 0.045), the number of involved vertebrae (P = 0.021) and lumbar location (P = 0.012) were statistically significant risk factors for major complication. When all above risk factors were incorporated in multivariate analysis, only the combined approach (P = 0.052) still remained significant. CONCLUSIONS: En bloc resection and anterior column reconstruction is accompanied by a high incidence of complications, especially when a combined approach is necessary.

The ghrelin receptor GHSR has two efficient agonists in the lobe-finned fish Latimeria chalumnae.

The peptide hormone ghrelin (an agonist) and LEAP2 (an antagonist) play important functions in energy metabolism via their receptor GHSR, an A-class G protein-coupled receptor. Ghrelin, LEAP2, and GHSR are widely present from fishes to mammals. However, our recent study suggested that fish GHSRs have different binding properties to ghrelin: a GHSR from the lobe-finned fish Latimeria chalumnae (coelacanth) is efficiently activated by ghrelin, but GHSRs from the ray-finned fish Danio rerio (zebrafish) and Larimichthys crocea (large yellow croaker) have lost binding to ghrelin. Do fish GHSRs use another peptide as their agonist? In the present study we tested to two fish motilins from D. rerio and L. chalumnae because motilin is distantly related to ghrelin. In ligand binding and activation assays, the fish GHSRs from D. rerio and L. crocea displayed no detectable or very low binding to all tested motilins; however, the fish GHSR from L. chalumnae bound to its motilin with high affinity and was efficiently activated by it. Therefore, it seemed that motilin is not a ligand for GHSR in the ray-finned fish D. rerio and L. crocea, but is an efficient agonist for GHSR in the lobe-finned fish L. chalumnae, one of the closest fish relatives of tetrapods. The results of present study suggested that GHSR might have two efficient agonists, ghrelin and motilin, in ancient fishes; however, this feature might be only preserved in some extant fishes with ancient evolutionary origins.

Nanomolar range of FAM237B can activate receptor GPR83.

Our recent study confirmed that the mature neuropeptide FAM237A, also known as neurosecretory protein GL (NPGL), is an efficient agonist for GPR83. The paralog FAM237B was previously reported as a weak agonist for GPR83. In the present study, we prepared mature human FAM237B via an intein-fusion approach and demonstrated that it could cause a significant activation effect at the nanomolar range (1‒10 nM) in a NanoBiT-based ß-arrestin recruitment assay. Thus, FAM237B appears to be another endogenous agonist for GPR83 and future in vivo studies will be required to confirm this.

Inhibiting Polyamide Intrusion of Thin Film Composite Membranes: Strategies and Environmental Implications.

Thin film composite polyamide (TFC) nanofiltration (NF) membranes represent extensive applications at the water-energy-environment nexus, which motivates unremitting efforts to explore membranes with higher performance. Intrusion of polyamide into substrate pores greatly restricts the overall membrane permeance because of the excessive hydraulic resistance, while the effective inhibition of intrusion remains technically challenging. Herein, we propose a synergetic regulation strategy of pore size and surface chemical composition of the substrate to optimize selective layer structure, achieving the inhibition of polyamide intrusion effective for the membrane separation performance enhancement. Although reducing the pore size of the substrate prevented polyamide intrusion at the intrapore, the membrane permeance was adversely affected due to the exacerbated "funnel effect". Optimizing the polyamide structure via surface chemical modification of the substrate, where reactive amino sites were in situ introduced by the ammonolysis of polyethersulfone substrate, allowed for maximum membrane permeance without reducing the substrate pore size. The optimal membrane exhibited excellent water permeance, ion selectivity, and emerging contaminants removal capability. The accurate optimization of selective layer is anticipated to provide a new avenue for the state-of-the-art membrane fabrication, which opens opportunities for promoting more efficient membrane-based water treatment applications.

Nucleobase Clustering Contributes to the Formation and Hollowing of Repeat-Expansion RNA Condensate.

RNA molecules with repeat expansion sequences can phase separate into gel-like condensate, which could lead to neurodegenerative diseases. Here, we report that, in the presence of Mg2+, RNA molecules containing 20× CAG repeats self-assemble into three morphologically distinct droplets. Using hyperspectral stimulated Raman microscopy, we show that RNA phase separation is accompanied by the clustering of nucleobases while forfeiting the canonical base-paired structure. As the RNA/Mg2+ ratio increases, the RNA droplets first expand and then shrink to adopt hollow vesicle-like structures. Significantly, for both large and vesicle-like RNA droplets, the nucleobase-clustered structure is more prominent at the rim, suggesting a continuously hardening process. This mechanism may be implicated in the general aging processes of RNA-containing membrane-less organelles.

LPAR5 confers radioresistance to cancer cells associated with EMT activation via the ERK/Snail pathway.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a critical event contributing to more aggressive phenotypes in cancer cells. EMT is frequently activated in radiation-targeted cells during the course of radiotherapy, which often endows cancers with acquired radioresistance. However, the upstream molecules driving the signaling pathways of radiation-induced EMT have not been fully delineated. METHODS: In this study, RNA-seq-based transcriptome analysis was performed to identify the early responsive genes of HeLa cells to γ-ray irradiation. EMT-associated genes were knocked down by siRNA technology or overexpressed in HeLa cells and A549 cells, and the resulting changes in phenotypes of EMT and radiosensitivity were assessed using qPCR and Western blotting analyses, migration assays, colony-forming ability and apoptosis of flow cytometer assays. RESULTS: Through RNA-seq-based transcriptome analysis, we found that LPAR5 is downregulated in the early response of HeLa cells to γ-ray irradiation. Radiation-induced alterations in LPAR5 expression were further revealed to be a bidirectional dynamic process in HeLa and A549 cells, i.e., the early downregulating phase at 2 ~ 4 h and the late upregulating phase at 24 h post-irradiation. Overexpression of LPAR5 prompts EMT programing and migration of cancer cells. Moreover, increased expression of LPAR5 is significantly associated with IR-induced EMT and confers radioresistance to cancer cells. Knockdown of LPAR5 suppressed IR-induced EMT by attenuating the activation of ERK signaling and downstream Snail, MMP1, and MMP9 expression. CONCLUSIONS: LPAR5 is an important upstream regulator of IR-induced EMT that modulates the ERK/Snail pathway. This study provides further insights into understanding the mechanism of radiation-induced EMT and identifies promising targets for improving the effectiveness of cancer radiation therapy.

Direct Imaging of Integrated Circuits in CPU with 60 nm Super-Resolution Optical Microscope.

Far-field super-resolution optical microscopies have achieved incredible success in life science for visualization of vital nanostructures organized in single cells. However, such resolution power has been much less extended to material science for inspection of human-made ultrafine nanostructures, simply because the current super-resolution optical microscopies modalities are rarely applicable to nonfluorescent samples or unlabeled systems. Here, we report an antiphase demodulation pump-probe (DPP) super-resolution microscope for direct optical inspection of integrated circuits (ICs) with a lateral resolution down to 60 nm. Because of the strong pump-probe (PP) signal from copper, we performed label-free super-resolution imaging of multilayered copper interconnects on a small central processing unit (CPU) chip. The label-free super-resolution DPP optical microscopy opens possibilities for easy, fast, and large-scale electronic inspection in the whole pipeline chain for designing and manufacturing ICs.

The impact of global dimming on crop yields is determined by the source-sink imbalance of carbon during grain filling.

Global dimming reduces incident global radiation but increases the fraction of diffuse radiation, and thus affects crop yields; however, the underlying mechanisms of such an effect have not been revealed. We hypothesized that crop source-sink imbalance of either carbon (C) or nitrogen (N) during grain filling is a key factor underlying the effect of global dimming on yields. We presented a practical framework to assess both C and N source-sink relationships, using data of biomass and N accumulation from periodical sampling conducted in field experiments for wheat and rice from 2013 to 2016. We found a fertilization effect of the increased diffuse radiation fraction under global dimming, which alleviated the negative impact of decreased global radiation on source supply and sink growth, but the source supply and sink growth were still decreased by dimming, for both C and N. In wheat, the C source supply decreased more than the C sink demand, and as a result, crops remobilized more pre-heading C reserves, in response to dimming. However, these responses were converse in rice, which presumably stemmed from the more increment in radiation use efficiency and the more limited sink size in rice than wheat. The global dimming affected source supply and sink growth of C more significantly than that of N. Therefore, yields in both crops were dependent more on the source-sink imbalance of C than that of N during grain filling. Our revealed source-sink relationships, and their differences and similarities between wheat and rice, provide a basis for designing strategies to alleviate the impact of global dimming on crop productivity.

Unusual orthologs shed new light on the binding mechanism of ghrelin to its receptor GHSR1a.

The gastric peptide ghrelin has important functions in energy metabolism and cellular homeostasis by activating growth hormone secretagogue receptor type 1a (GHSR1a). The N-terminal residues of ghrelin orthologs from all vertebrates are quite conserved; however, in orthologs from Cavia porcellus and Phyllostomus discolor, Ser2 and Leu5 are replaced by a smaller Ala and a positively charged Arg, respectively. In the present study, we first demonstrated that the hydrophobic Leu5 is essential for the function of human ghrelin, because Ala replacement caused an approximately 100-fold decrease in activity. However, replacement of Leu5 by an Arg residue caused much less disruption; further replacement of Ser2 by Ala almost restored full activity, although the [S2A] mutation itself showed slight detriments, implying that the positively charged Arg5 in the [S2A,L5R] mutant might form alternative interactions with certain receptor residues to compensate for the loss of the essential Leu5. To identify the responsible receptor residues, we screened GHSR1a mutants in which all conserved negatively charged residues in the extracellular regions and all aromatic residues in the ligand-binding pocket were mutated separately. According to the decrease in selectivity of the mutant receptors towards [S2A,L5R]ghrelin, we deduced that the positively charged Arg5 of the ghrelin mutant primarily interacts with the essential aromatic Phe286 at the extracellular end of the sixth transmembrane domain of GHSR1a by forming cation-π and π-π interactions. The present study provided new insights into the binding mechanism of ghrelin with its receptor, and thus would facilitate the design of novel ligands for GHSR1a.

LEAP2 has antagonized the ghrelin receptor GHSR1a since its emergence in ancient fish.

Recent studies have demonstrated that liver-expressed antimicrobial peptide 2 (LEAP2) antagonizes the ghrelin receptor GHSR1a in mammals. However, its antagonistic function in lower vertebrates has not yet been tested. LEAP2 orthologs have been identified from a variety of fish species; however, previous studies all focused on their antimicrobial activity. To test whether LEAP2 functions as a GHSR1a antagonist in the lowest vertebrates, we studied the antagonism of a fish LEAP2 from Latimeria chalumnae, an extant coelacanth that is one of the closest living fish relatives of tetrapods. Using binding assays, we demonstrated that the coelacanth LEAP2 and ghrelin bound to the coelacanth GHSR1a with IC50 values in the nanomolar range. Using activation assays, we demonstrated that the coelacanth ghrelin activated the coelacanth GHSR1a with an EC50 value in the nanomolar range, and this activation effect was efficiently antagonized by a nanomolar range of the coelacanth LEAP2. In addition, we also showed that the human LEAP2 and ghrelin were as effective as their coelacanth orthologs towards the coelacanth GHSR1a; however, the coelacanth peptides had moderately lower activity towards the human GHSR1a. Thus, LEAP2 serves as an endogenous antagonist of the ghrelin receptor GHSR1a in coelacanth and the ghrelin-LEAP2-GHSR1a system has evolved slowly since its emergence in ancient fish.

Identifying key residues and key interactions for the binding of LEAP2 to receptor GHSR1a.

Liver-expressed antimicrobial peptide 2 (LEAP2) was recently identified as a competitive antagonist for the G protein-coupled receptor GHSR1a, the cognate receptor for the gastric peptide ghrelin. LEAP2 plays important functions in energy metabolism by tuning the ghrelin-GHSR1a system. However, the molecular mechanism by which LEAP2 binds to GHSR1a is largely unknown. In the present study, we first conducted alanine-scanning mutagenesis on the N-terminal fragment of human LEAP2 and demonstrated that the positively charged Arg6 and the aromatic Phe4 are essential for LEAP2 binding to GHSR1a. To identify the receptor residues interacting with the essential Arg6 and Phe4 of LEAP2, we conducted extensive site-directed mutagenesis on GHSR1a. After all conserved negatively charged residues in the extracellular regions of human GHSR1a were mutated, only mutation of Asp99 caused much more detriments to GHSR1a binding to LEAP2 than binding to ghrelin, suggesting that the absolutely conserved Asp99 of GHSR1a probably interacts with the essential Arg6 of LEAP2. After five conserved Phe residues in the predicted ligand-binding pocket of human GHSR1a were mutated, three of them were identified as important for GHSR1a binding to LEAP2. According to a structural model of GHSR1a, we deduced that the adjacent Phe279 and Phe312 might interact with the essential Phe4 of LEAP2, while Phe119 might interact with the aromatic Trp5 of LEAP2. The present study provided new insights into the interaction of LEAP2 with its receptor, and would facilitate the design of novel ligands for GHSR1a in future studies.

Precise Encoding of Triple-Bond Raman Scattering of Single Polymer Nanoparticles for Multiplexed Imaging Application.

Stimulated Raman scattering (SRS) microscopy in combination with innovative tagging strategies offers great potential as a universal high-throughput biomedical imaging tool. Here, we report rationally tailored small molecular monomers containing triple-bond units with large Raman scattering cross-sections, which can be polymerized at the nanoscale for enhancement of SRS contrast with smaller but brighter optical nanotags with artificial fingerprint output. From this, a class of triple-bond rich polymer nanoparticles (NPs) was engineered by regulating the relative dosages of three chemically different triple-bond monomers in co-polymerization. The bonding strategy allowed for 15 spectrally distinguishable triple-bond combinations. These accurately structured nano molecular aggregates, rather than long-chain macromolecules, could establish a universal method for generating small-sized biological SRS imaging tags with high sensitivity for high-throughput multi-color biomedical imaging.

The acclimation of leaf photosynthesis of wheat and rice to seasonal temperature changes in T-FACE environments.

Crops show considerable capacity to adjust their photosynthetic characteristics to seasonal changes in temperature. However, how photosynthesis acclimates to changes in seasonal temperature under future climate conditions has not been revealed. We measured leaf photosynthesis (An ) of wheat (Triticum aestivum L.) and rice (Oryza sativa L.) grown under four combinations of two levels of CO2 (ambient and enriched up to 500 µmol/mol) and two levels of canopy temperature (ambient and increased by 1.5-2.0°C) in temperature by free-air CO2 enrichment (T-FACE) systems. Parameters of a biochemical C3 -photosynthesis model and of a stomatal conductance (gs ) model were estimated for the four conditions and for several crop stages. Some biochemical parameters related to electron transport and most gs parameters showed acclimation to seasonal growth temperature in both crops. The acclimation response did not differ much between wheat and rice, nor among the four treatments of the T-FACE systems, when the difference in the seasonal growth temperature was accounted for. The relationships between biochemical parameters and leaf nitrogen content were consistent across leaf ranks, developmental stages, and treatment conditions. The acclimation had a strong impact on gs model parameters: when parameter values of a particular stage were used, the model failed to correctly estimate gs values of other stages. Further analysis using the coupled gs -biochemical photosynthesis model showed that ignoring the acclimation effect did not result in critical errors in estimating leaf photosynthesis under future climate, as long as parameter values were measured or derived from data obtained before flowering.

Coherent Raman Scattering Unravelling Mechanisms Underlying Skull Optical Clearing for Through-Skull Brain Imaging.

Optical access of a mouse brain using microscopes is the key to study brain structures and functions in vivo. However, the opaque skull of a mouse has to be either opened or thinned in an invasive way to attain an adequate imaging depth in the brain. Mild skull optical clearing is highly desired, but its chemical mechanism is far from being understood. Here, we unraveled the molecular process underlying optical clearing of the mouse skull by label-free hyperspectral stimulated Raman scattering (SRS) microscopy, thereby discovering the optimal clearing strategy to turn a turbid skull into a transparent skull window. Furthermore, we demonstrated in vivo three-photon imaging of vascular structures as deep as 850 µm in the cortex of the mouse brain. Coherent Raman based microspectroscopy holds great promise to advance skull and tissue clearing methods in the future.

Development of a novel ligand binding assay for relaxin family peptide receptor 3 and 4 using NanoLuc complementation.

Relaxin family peptides perform a variety of biological functions by binding and activating relaxin family peptide receptor 1-4 (RXFP1-4), four A-class G protein-coupled receptors. In the present work, we developed a novel ligand binding assay for RXFP3 and RXFP4 based on NanoLuc complementation technology (NanoBiT). A synthetic ligation version of the low-affinity small complementation tag (SmBiT) was efficiently ligated to the A-chain N terminus of recombinant chimeric agonist R3/I5 using recombinant circular sortase A. After the ligation product R3/I5-SmBiT was mixed with human RXFP3 or RXFP4 genetically fused with a secretory large NanoLuc fragment (sLgBiT) at the N terminus, NanoLuc complementation was induced by high-affinity ligand-receptor binding. Binding kinetics and affinities of R3/I5-SmBiT with sLgBiT-fused RXFP3 and RXFP4 were conveniently measured according to the complementation-induced bioluminescence. Using R3/I5-SmBiT and the sLgBiT-fused receptor as a complementation pair, binding potencies of various ligands with RXFP3 and RXFP4 were quantitatively measured without the cumbersome washing step. The novel NanoBiT-based ligand binding assay is convenient for use and suitable for automation, thus will facilitate interaction studies of RXFP3 and RXFP4 with ligands in future. This assay can also be applied to some other plasma membrane receptors for pharmacological characterization of ligands in future studies.

Rehabilitation increases cortical activation during single-leg stance in patients with chronic ankle instability.

Background: Chronic ankle instability (CAI) has been considered a neurophysiological disease, having as symptoms dysfunction in somatosensory and motor system excitability. Rehabilitation has been considered an effective treatment for CAI. However, few studies have explored the effects of rehabilitation on neuroplasticity in the CAI population. Objective: The purpose of this study was to investigate the effects of rehabilitation on cortical activities for postural control in CAI patients and to find the correlation between the change in cortical activities and patient-reported outcomes (PROs). Methods: Thirteen participants with CAI (6 female, 7 male, age = 33.8 ± 7.7 years, BMI = 24.7 ± 4.9 kg/m2) received a home exercise program for about 40 min per day, four days per week and six weeks, including ankle range-of-motion exercise, muscle strengthening, and balance activities. Cortical activation, PROs and Y-balance test outcomes were assessed and compared before and after rehabilitation. Cortical activation was detected via Functional near-infrared spectroscopy (fNIRS) while the participants performed single-leg stance tasks. Results: The participants had better PROs and Y balance test outcomes after rehabilitation. Greater cortical activation was observed in the primary somatosensory cortex (S1, d = 0.66, p = 0.035), the superior temporal gyrus (STG, d = 1.06, p = 0.002) and the middle temporal gyrus (MTG, d = 0.66, p = 0.035) in CAI patients after rehabilitation. Moreover, significant positive correlations were observed between the recovery of ankle symptoms and the change of cortical activation in S1 (r = 0.74, p = 0.005) and STG (r = 0.72, p = 0.007) respectively. Conclusion: The current study reveals that six weeks of rehabilitation can cause greater cortical activation in S1, STG and MTG. This increase in cortical activation suggested a better ability to perceive somatosensory stimuli and may have a compensatory role in function improvement.

An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase.

In recent years, a few asparaginyl endopeptidases (AEPs) from certain higher plants have been identified as efficient peptide ligases with wide applications in protein labeling and cyclic peptide synthesis. Recently, we developed a NanoLuc Binary Technology (NanoBiT)-based peptide ligase activity assay to identify more AEP-type peptide ligases. Herein, we screened 61 bamboo species from 16 genera using this assay and detected AEP-type peptide ligase activity in the crude extract of all tested bamboo leaves. From a popular bamboo species, Bambusa multiplex, we identified a full-length AEP-type peptide ligase candidate (BmAEP1) via transcriptomic sequencing. After its zymogen was overexpressed in Escherichia coli and self-activated in vitro, BmAEP1 displayed high peptide ligase activity, but with considerable hydrolytic activity. After site-directed mutagenesis of its ligase activity determinants, the mutant zymogen of [G238V]BmAEP1 was normally overexpressed in E. coli, but failed to activate itself. To resolve this problem, we developed a novel protease-assisted activation approach in which trypsin was used to cleave the mutant zymogen and was then conveniently removed via ion-exchange chromatography. After the noncovalently bound cap domain was dissociated from the catalytic core domain under acidic conditions, the recombinant [G238V]BmAEP1 displayed high peptide ligase activity with much lower hydrolytic activity and could efficiently catalyze inter-molecular protein ligation and intramolecular peptide cyclization. Thus, the engineered bamboo-derived peptide ligase represents a novel tool for protein labeling and cyclic peptide synthesis.

Diverse applications and development of aptamer detection technology.

Aptamers have received extensive attention in recent years because of their advantages of high specificity, high sensitivity and low immunogenicity. Aptamers can perform almost all functions of antibodies through the combination of spatial structure and target, which are called "chemical antibodies". At present, aptamers have been widely used in cell imaging, new drug development, disease treatment, microbial detection and other fields. Due to the diversity of modifications, aptamers can be combined with different detection technologies to construct aptasensors. This review focuses on the diversity of aptamers in the field of detection and the development of aptamer-based detection technology and proposes new challenges for aptamers in this field.