Template-controllable rolling circle amplification for dual protein sensitive analysis.

Conjoint analysis of multiple protein biomarkers can improve the accuracy of disease analysis. Rolling circle amplification (RCA) generates different products by designing circular templates, which can subsequently bind with specific probes to generate various fluorescence signals; thus, it has potential for application in the analysis of various protein biomarkers. Current RCA approaches to detect proteins commonly follow an indirect primer-controlled RCA mode. And the molecular beacon probe combines with RCA products through free collision to generate signals, resulting in lower reaction efficiency. Herein, we propose a direct template-controlled RCA mode using nanosheets as carriers and quenchers for fluorescent probes to simultaneously detect two protein biomarkers. A dual functional magnetic bead was first designed to recognize and capture two proteins while releasing two templates for subsequent RCA. RCA products compete with probes loaded on two-dimensional metal-organic framework nanosheets for hybridization, completing the transition from single-stranded to double-stranded DNA. Double-stranded DNA is far from the nanosheets, and the recovered fluorescence signal can be used to evaluate the concentration of target proteins. This method exhibits excellent analytical performance and can successfully achieve the analysis of Tau and AßO in Alzheimer's disease clinical cerebrospinal fluid samples.

Magneto-assisted enzymatic DNA walkers for simultaneous electrochemical detection of amyloid-beta oligomers and Tau.

DNA walkers have been widely explored and applied as biosensor elements to detect disease-related biomarkers. Traditional interface-anchored DNA walkers typically have a fixed swing arm range and an orientation of the preset track, which might complicate the design of a sensor system and limit its application in more scenes. We propose a simple electrochemical aptasensor to accurately detect Alzheimer's disease (AD) based on a nicking enzyme-powered DNA walker. In this method, bifunctional magnetic nanoparticles are used to identify and capture Aß oligomers (AßO) and Tau and release the DNA walker. As the DNA walker moves freely on the surface of the electrode, the nicking enzymes circularly cleave and release the two signal substrate chains, significantly amplifying the signal. It has been demonstrated that the constructed sensor can sensitively detect AßO and Tau, and the combined analysis of dual markers improves the accuracy of AD diagnosis. Furthermore, this method can distinguish normal individuals from AD patients in real cerebrospinal fluid samples. The excellent performance of this biosensor makes it promising for clinical applications in diagnosing AD patients and prognosis assessment.

Assembly of DNA triangular pyramid frustum for ultrasensitive quantification of exosomal miRNA.

Early screening of biomarkers benefits therapy and prognosis of cancers. MiRNAs encapsulated in tumor-derived exosomes are emerging biomarkers for early diagnosis of cancers. Nevertheless, traditional methods suffer certain drawbacks, which hamper their wide applications. In this contribution, we have developed a convenient electrochemical approach for quantification of exosomal miRNA based on the assembly of DNA triangular pyramid frustum (TPF) and strand displacement amplification. Four single-stranded DNA helps the formation of primary DNA triangle with three thiols for gold electrode immobilization at the bottom and three amino groups on overhangs for the capture of silver nanoparticles. On the other hand, target miRNA induced strand displacement reaction produces abundant specific DNA strands, which help the DNA structural transition from triangle to TPF. Amino groups are thus hidden and the declined silver stripping current can be used for the evaluation of target miRNA concentration. This biosensor exhibits excellent analytical performances and successfully achieves analysis of exosomal miRNAs from cells and clinical serum samples.

Silencing of the calcium-dependent protein kinase TaCDPK27 improves wheat resistance to powdery mildew.

BACKGROUND: Calcium ions (Ca2+), secondary messengers, are crucial for the signal transduction process of the interaction between plants and pathogens. Ca2+ signaling also regulates autophagy. As plant calcium signal-decoding proteins, calcium-dependent protein kinases (CDPKs) have been found to be involved in biotic and abiotic stress responses. However, information on their functions in response to powdery mildew attack in wheat crops is limited. RESULT: In the present study, the expression levels of TaCDPK27, four essential autophagy-related genes (ATGs) (TaATG5, TaATG7, TaATG8, and TaATG10), and two major metacaspase genes, namely, TaMCA1 and TaMCA9, were increased by powdery mildew (Blumeria graminis f. sp. tritici, Bgt) infection in wheat seedling leaves. Silencing TaCDPK27 improves wheat seedling resistance to powdery mildew, with fewer Bgt hyphae occurring on TaCDPK27-silenced wheat seedling leaves than on normal seedlings. In wheat seedling leaves under powdery mildew infection, silencing TaCDPK27 induced excess contents of reactive oxygen species (ROS); decreased the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT); and led to an increase in programmed cell death (PCD). Silencing TaCDPK27 also inhibited autophagy in wheat seedling leaves, and silencing TaATG7 also enhanced wheat seedling resistance to powdery mildew infection. TaCDPK27-mCherry and GFP-TaATG8h colocalized in wheat protoplasts. Overexpressed TaCDPK27-mCherry fusions required enhanced autophagy activity in wheat protoplast under carbon starvation. CONCLUSION: These results suggested that TaCDPK27 negatively regulates wheat resistance to PW infection, and functionally links with autophagy in wheat.

Tracing cellular interaction of circRNA-miRNA axis with Cu metal-organic framework supported DNA cascade assembly.

Circular RNAs (circRNAs) can act as molecular sponges of microRNA (miRNA) to form circRNA-miRNA axis, which regulates the expressions of downstream proteins. Although the mechanism has been widely reported in various bioprocesses, there is still a lack of reliable and facile way to intuitively monitor and locate the interaction between circRNA and miRNA inside living cells. In this study, multiple DNA probes are designed and loaded onto two-dimensional Cu metal-organic framework (2D Cu-MOF) nanosheets for one-step analysis of circRNA-miRNA axis. The nanosheets serve as not only powerful fluorescence quenchers but also excellent nanocarriers of abundant DNA probes for further assembly. The Probes@Cu-MOF complex can be applied to track the circRNA-miRNA axis in living cells with high sensitivity and co-localization analysis. This platform combines the transmembrane advantage of nanosheets and the signal amplification ability of localized DNA cascade assembly, so it holds great potential for understanding the biological functions of circRNA-miRNAs in cancer pathogenesis and for therapeutic applications.

Immunotherapeutic approach to reduce senescent cells and alleviate senescence-associated secretory phenotype in mice.

Accumulation of senescent cells (SNCs) with a senescence-associated secretory phenotype (SASP) has been implicated as a major source of chronic sterile inflammation leading to many age-related pathologies. Herein, we provide evidence that a bifunctional immunotherapeutic, HCW9218, with capabilities of neutralizing TGF-ß and stimulating immune cells, can be safely administered systemically to reduce SNCs and alleviate SASP in mice. In the diabetic db/db mouse model, subcutaneous administration of HCW9218 reduced senescent islet ß cells and SASP resulting in improved glucose tolerance, insulin resistance, and aging index. In naturally aged mice, subcutaneous administration of HCW9218 durably reduced the level of SNCs and SASP, leading to lower expression of pro-inflammatory genes in peripheral organs. HCW9218 treatment also reverted the pattern of key regulatory circadian gene expression in aged mice to levels observed in young mice and impacted genes associated with metabolism and fibrosis in the liver. Single-nucleus RNA Sequencing analysis further revealed that HCW9218 treatment differentially changed the transcriptomic landscape of hepatocyte subtypes involving metabolic, signaling, cell-cycle, and senescence-associated pathways in naturally aged mice. Long-term survival studies also showed that HCW9218 treatment improved physical performance without compromising the health span of naturally aged mice. Thus, HCW9218 represents a novel immunotherapeutic approach and a clinically promising new class of senotherapeutic agents targeting cellular senescence-associated diseases.

Clinical effects of Chemotherapy combined with Immunotherapy in patients with advanced NSCLC and the effect on their nutritional status and immune function.

Objectives: To evaluate the clinical effects of chemotherapy combined with immunotherapy in patients with advanced non-small-cell lung cancer (NSCLC) and the effect on their nutritional status and immune function. Methods: Total 120 patients with advanced NSCLC admitted to Affiliated Hospital of Hebei University from May 2019 to October 2021 were randomly divided into two groups (n= 60, respectively). Patients in the control group were treated by chemotherapy with cisplatin-paclitaxel (TP) alone: 120 mg/m2 paclitaxel was used on d1; and 25mg/m2 cisplatin (CDDP) was used for more than two hour, once every 14 days, for three consecutive three cycles. Patients in the study group were additionally given 200 mg sindilizumab by intravenous drip, once every three weeks. The contrastive analysis of clinical effects, the incidence of adverse reactions, improvement of the nutrient index and the changes in levels of CD3+, CD4+, CD8+, and CD4+/CD8+ in T-lymphocyte subsets was performed between the two groups. Result: The overall response rate (ORR) was 80% and 61% in the study group and the control group, respectively; and the difference was statistically significant (p=0.03); the contrast analysis of the incidence of post-treatment adverse drug reactions (ADRs) in patients in the two groups suggested that the incidence of adverse reactions was 33.3% and 45% in the study group and the control group, respectively; and the difference was not statistically significant (p=0.19). After the treatment, the improvement of hemoglobin, albumin, serum iron and ferritin levels in the study group was more significant than that in the control group; and the difference was statistically significant (p < 0.05). After the treatment, the levels of CD3+, CD4+ and CD4+/CD8+ in the study group were much higher than those in the control group; and the difference was statistically significant (p < 0.05). Conclusion: Chemotherapy combined with immunotherapy is effective in treating patients with advanced NSCLC without increasing the incidence of adverse reactions, and can significantly improve their nutritional status and T-lymphocyte function. This therapeutic regimen is of much higher clinical value than the chemotherapy-only regimen.

Multivalent aptamer nanoscaffold cytosensor for glioma circulating tumor cells during Epithelial-Mesenchymal transition.

The key factor that causes glioma invasion and metastasis is circulating tumor cells (CTCs) undergoing epithelial-mesenchymal transition (EMT). Effective analysis of EMT-CTCs can provide an important foundation for early detection and prognosis monitoring of glioma, but the changes in the biomarkers of CTCs in different states of EMT make detection difficult. In this study, we developed a multivalent aptamer nanoscaffold-based electrochemical cytosensor (MAS-cytosensor) to efficiently detect EMT-CTCs. The two chains forming the MAS are composed of a specific aptamer detector, a binding region for DNA self-assembly, and a foothold for interface anchoring. When target CTCs exist, the bisaptamer detector on MAS can sensitively identify CTCs and pull them to the electrode surface, generating electrochemical signals. It has been demonstrated that the MAS-cytosensor can not only detect EMT-CTCs sensitively (detection limit of 6 cells/mL in buffer), but also allows for further downstream analysis after release with high viability. Overall, this cytosensor provides a reliable detection solution for CTCs regardless of their EMT status, and provides an efficient method for in-depth study role of the post-EMT CTCs in clinical application and metastasis mechanisms.

Heteromultivalent scaffolds fabricated by biomimetic co-assembly of DNA-RNA building blocks for the multi-analysis of miRNAs.

Heteromultivalent scaffolds with different repeated monomers have great potential in biomedicine, but convenient construction strategies for integrating various functional modules to achieve multiple biological functions are still lacking. Here, taking advantage of the heteromultivalent effect of dendritic nucleic acids and the specific biochemical properties of microRNAs (miRNAs), we assembled novel heteromultivalent nucleic acid scaffolds by biomimetic co-assembly of DNA-RNA building blocks. In our approach, two miRNAs were used to initiate and maintain dendritic structures in an interdependent manner; so, the heteromultivalent nanostructure can only form in the presence of both miRNAs. The proposed nanostructure can be used for one-step analysis of two miRNAs in an AND logic format. Taking miR-18b-5p and miR-342-3p which are associated with Alzheimer's disease as an example, a FRET sensing system was fabricated for the simultaneous analysis of two miRNAs within one hour at picomolar concentration. Further studies show that the designed device may have the potential to distinguish between AD patients and the healthy population by analysis of two miRNAs in CSF (cerebrospinal fluid) samples, suggesting its possible applicability in clinics.

Highly Sensitive Aptasensor for Detecting Cancerous Exosomes Based on Clover-like Gold Nanoclusters.

Exosome-based liquid biopsy technologies play an increasingly prominent role in tumor diagnosis. However, the simple and sensitive method for counting exosomes still faces considerable challenges. In this work, the CD63 aptamer-modified DNA tetrahedrons on the gold electrode were used as recognition elements for the specific capture of exosomes. Partially complementary DNA probes act as bridges linking trapped exosomes and three AuNP-DNA signal probes. This clover-like structure can tackle the recognition and sensitivity issues arising from the undesired AuNP aggregation event. When cancerous exosomes are present in the system, the high accumulation of methylene blue molecules from DNA-AuNP nanocomposites on the surface of the electrode leads to an intense current signal. According to the results, the aptasensor responds to MCF-7 cell-derived exosomes in the concentration range from 1.0 × 103 to 1.0 × 108 particles·µL-1, with the detection limit of 158 particles·µL-1. Furthermore, the aptasensor has been extended to serum samples from breast cancer patients and exhibited excellent specificity. To sum it up, the aptasensor is sensitive, straightforward, less expensive, and fully capable of receiving widespread application in clinics for tumor monitoring.

In vitro and in vivo activity of meropenem+avibactam against MBL-producing carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: Antibiotic resistance has become a public health problem to be solved worldwide and metallo-ß-lactamase (MBL)-producing bacteria make this problem even more challenging. METHODS: The interactions of meropenem (MEM) in combination with avibactam (AVI) in growth inhibition on MBL-producing carbapenem-resistant Klebsiella pneumoniae (CRKP) strains were tested. In vitro interactions of MEM+AVI were tested using the microdilution checkerboard assay and time-kill curves. In vivo interactions of MEM+AVI were tested using the Galleria mellonella model. RESULTS: All strains were multi-drug resistant strains and six of them were proved to produce MBLs. We show that the combination of MEM+AVI generates profound synergistic effects on growth inhibition of all strains, which was better than that of MEM+vaborbactam or imipenem+relebactam. The time-kill curves further confirmed the potent synergistic antibacterial effects of MEM+AVI against MBL-producing CRKP strains. Galleria mellonella studies were consistent with in vitro analysis. Combining MEM with AVI improved survival rates and mean survival days were obviously prolonged compared to the drug alone and the untreated controls. CONCLUSIONS: To our knowledge, this study is the first report of MEM+AVI collaborating against MBL-producing CRKP strains. Our findings showed that the combination of MEM+AVI has the potential for antibiotic drug development to combat MBL-producing pathogens.

DNA-functionalized gold nanoparticles: Modification, characterization, and biomedical applications.

With the development of technologies based on gold nanoparticles (AuNPs), bare AuNPs cannot meet the increasing requirements of biomedical applications. Modifications with different functional ligands are usually needed. DNA is not only the main genetic material, but also a good biological material, which has excellent biocompatibility, facile design, and accurate identification. DNA is a perfect ligand candidate for AuNPs, which can make up for the shortcoming of bare AuNPs. DNA-modified AuNPs (DNA-AuNPs) have exciting features and bright prospects in many fields, which have been intensively investigated in the past decade. In this review, we summarize the various approaches for the immobilization of DNA strands on the surface of AuNPs. Representative studies for biomedical applications based on DNA-AuNPs are also discussed. Finally, we present the challenges and future directions.

Clinical Profile and Risk Factors of Group B Streptococcal Colonization in Mothers from the Eastern District of China.

Objective: The main aim of this study was to determine the prevalence, capsular genotyping, antimicrobial susceptibility, and associated factors of colonizing Group B Streptococcus (GBS) in pregnant women admitted to a hospital in Jinan, East China. Methods: Demographic data, clinical characteristics, and vaginal and rectal swabs were obtained from a group of expecting mothers subjected to GBS screening at the late stage of pregnancy who went into labor over the period from November 2019 to October 2020. Identification of GBS and determination of antimicrobial resistance patterns were performed using a BD Phoenix-100 system. Capsular genotypes were analyzed using polymerase chain reaction and the associated factors were evaluated via logistic regression. Result: A total of 2761 pregnant women were recruited for this study. The GBS colonization rate was 6.70% (185/2761). Among the 172 GBS strains examined, all were susceptible to vancomycin and linezolid. Resistance was the highest for erythromycin (80.2%), followed by clindamycin (75.0%), levofloxacin (65.1%), and tetracycline (57.6%). The most common serotype identified was Ia (61.0%), followed by III (29.7%), VI (4.6%), II (3.5%), VII (0.6%), and a nontypeable strain. Risk factors for maternal GBS colonization included maternal age (older than 30 years) (OR = 1.913 (1.662, 2.478)), gestational age at birth (average gestational age) (OR = 1.992 (1.445, 2.746)), and prelabor rupture of membrane (OR = 3.838 (1.619, 9.099)). Conclusion: The prevalence of GBS was relatively low. The maternal age was a factor associated with GBS colonization. Subjects showing GBS positivity during late pregnancy were prone to prolonged rupture of the membrane (PROM) and birth at lower a gestation age than the GBS-negative group. Penicillin could still be used as the first agent of choice for intrapartum antibiotic prophylaxis (IAP).

Drug development concerning metallo-ß-lactamases in gram-negative bacteria.

ß-Lactams have been a clinical focus since their emergence and indeed act as a powerful tool to combat severe bacterial infections, but their effectiveness is threatened by drug resistance in bacteria, primarily by the production of serine- and metallo-ß-lactamases. Although once of less clinical relevance, metallo-ß-lactamases are now increasingly threatening. The rapid dissemination of resistance mediated by metallo-ß-lactamases poses an increasing challenge to public health worldwide and comprises most existing antibacterial chemotherapies. Regrettably, there have been no clinically available inhibitors of metallo-ß-lactamases until now. To cope with this unique challenge, researchers are exploring multidimensional strategies to combat metallo-ß-lactamases. Several studies have been conducted to develop new drug candidates or calibrate already available drugs against metallo-ß-lactamases. To provide an overview of this field and inspire more researchers to explore it further, we outline some promising candidates targeting metallo-ß-lactamase producers, with a focus on Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. Promising candidates in this review are composed of new antibacterial drugs, non-antibacterial drugs, antimicrobial peptides, natural products, and zinc chelators, as well as their combinations with existing antibiotics. This review may provide ideas and insight for others to explore candidate metallo-ß-lactamases as well as promote the improvement of existing data to obtain further convincing evidence.

Fear of recurrence in elderly patients with coronary heart disease: the current situation and influencing factors according to a questionnaire analysis.

OBJECTIVE: Fear of recurrence is a common psychosocial sequela among patients with heart disease. Analyses of coronary heart disease, particularly in elderly patients, are relatively rare. This study aimed to investigate the current situation in this context, as well as the influencing fear factors concerning recurrence in elderly patients with coronary heart disease. METHODS: A total of 200 elderly outpatients with coronary heart disease were recruited to participate in this survey from a tertiary hospital in Baoding (China). The questionnaires included items from the Disease Progression Simplified Scale, the Simplified Coping Style Questionnaire, and the Social Support Rating Scale (SSRS). Univariate and multivariate regression analyses were adopted to investigate the influencing factors on the fear of recurrence. RESULTS: The fear of recurrence score in elderly patients with coronary heart disease was (38.46 ± 8.13), among which 119 cases (59.5%) scored higher than 34 points. The SSRS total average score was (34.89 ± 9.83) points. Positive coping style and social support were negatively correlated with the total score of recurrence fear (r = - 0.621, - 0.413, both P < 0.001). There was a positive correlation between negative coping style and the total score of recurrence fear (r = 0.232, P < 0.001). Multiple linear regression analysis showed that the course of the disease, the number of disease recurrence cases, active coping, and social support were relevant factors in fear of recurrence (all P < 0.05). CONCLUSION: The detection rate of fear of recurrence in elderly patients with coronary heart disease was relatively high but could be reduced by active interventions and enhancing social support.

PD-L1 expression, tumor mutational burden, and immune cell infiltration in non-small cell lung cancer patients with epithelial growth factor receptor mutations.

Background: Immunotherapy using programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) inhibitors seems less effective in non-small cell lung cancer (NSCLC) patients with epithelial growth factor receptor (EGFR) mutations. Varied responses to PD-1/PD-L1 inhibitors have recently been observed in NSCLC patients harboring different types of EGFR mutations. Some EGFR-mutated NSCLC patients may benefit from PD-1/PD-L1 inhibitors. At present, PD-L1 expression, tumor mutational burden (TMB), and tumor immune microenvironment (TIME) are biomarkers for predicting the efficacy of PD-1/PD-L1 inhibitors in NSCLC patients. We retrospectively evaluated PD-L1 expression, TMB, and immune cell infiltration in NSCLC patients with EGFR mutation subtypes. Methods: PD-L1 expression, TMB, and the abundance of immune cell infiltration in NSCLC patients were evaluated in public databases and clinical samples. TMB was detected using the NGS technique, PD-L1 was detected using immunohistochemistry, and the abundance of immune cell infiltration in NSCLC samples was detected using multiple immunohistochemistry. Results: PD-L1 expression and TMB were lower in EGFR-mutated NSCLCs than in wild-type patients. Differences in the abundance of immune cell infiltration were also observed between EGFR-mutated and wild-type NSCLC. The expression of PD-L1, TMB, and abundance of immune cell infiltration were different in patients harboring different subtypes of EGFR mutations. Patients with uncommon EGFR mutations, especially the G719X mutation, showed higher TMB and expressions of PD-L1 than classical EGFR mutations. M1 macrophages were higher in uncommon EGFR mutations than classical EGFR mutations. Conclusions: The expression of PD-L1 and TMB in uncommon EGFR-mutated NSCLCs, especially the G719X mutation, were higher than those for classical EGFR-mutated NSCLCs and similar to EGFR wild-type. The abundance of immune cell infiltration in uncommon EGFR-mutated NSCLCs was similar to that in EGFR wild-type. Our findings suggest that uncommon EGFR-mutated NSCLCs may benefit from PD-1/PD-L1 inhibitors.

Dietary selection of metabolically distinct microorganisms drives hydrogen metabolism in ruminants.

Ruminants are important for global food security but emit the greenhouse gas methane. Rumen microorganisms break down complex carbohydrates to produce volatile fatty acids and molecular hydrogen. This hydrogen is mainly converted into methane by archaea, but can also be used by hydrogenotrophic acetogenic and respiratory bacteria to produce useful metabolites. A better mechanistic understanding is needed on how dietary carbohydrates influence hydrogen metabolism and methanogenesis. We profiled the composition, metabolic pathways, and activities of rumen microbiota in 24 beef cattle adapted to either fiber-rich or starch-rich diets. The fiber-rich diet selected for fibrolytic bacteria and methanogens resulting in increased fiber utilization, while the starch-rich diet selected for amylolytic bacteria and lactate utilizers, allowing the maintenance of a healthy rumen and decreasing methane production (p < 0.05). Furthermore, the fiber-rich diet enriched for hydrogenotrophic methanogens and acetogens leading to increased electron-bifurcating [FeFe]-hydrogenases, methanogenic [NiFe]- and [Fe]-hydrogenases and acetyl-CoA synthase, with lower dissolved hydrogen (42%, p < 0.001). In contrast, the starch-rich diet enriched for respiratory hydrogenotrophs with greater hydrogen-producing group B [FeFe]-hydrogenases and respiratory group 1d [NiFe]-hydrogenases. Parallel in vitro experiments showed that the fiber-rich selected microbiome enhanced acetate and butyrate production while decreasing methane production (p < 0.05), suggesting that the enriched hydrogenotrophic acetogens converted some hydrogen that would otherwise be used by methanogenesis. These insights into hydrogen metabolism and methanogenesis improve understanding of energy harvesting strategies, healthy rumen maintenance, and methane mitigation in ruminants.

The Metacaspase TaMCA-Id Negatively Regulates Salt-Induced Programmed Cell Death and Functionally Links With Autophagy in Wheat.

Metacaspases (MCAs), a family of caspase-like proteins, are important regulators of programmed cell death (PCD) in plant defense response. Autophagy is an important regulator of PCD. This study explored the underlying mechanism of the interaction among PCD, MCAs, and autophagy and their impact on wheat response to salt stress. In this study, the wheat salt-responsive gene TaMCA-Id was identified. The open reading frame (ORF) of TaMCA-Id was 1,071 bp, coding 356 amino acids. The predicted molecular weight and isoelectric point were 38,337.03 Da and 8.45, respectively. TaMCA-Id had classic characteristics of type I MCAs domains, a typical N-terminal pro-domain rich in proline. TaMCA-Id was mainly localized in the chloroplast and exhibited nucleocytoplasmictrafficking under NaCl treatment. Increased expression of TaMCA-Id in wheat seedling roots and leaves was triggered by 150 mM NaCl treatment. Silencing of TaMCA-Id enhanced sensitivity of wheat seedlings to NaCl stress. Under NaCl stress, TaMCA-Id-silenced seedlings exhibited a reduction in activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), higher accumulation of H2O2 and O 2 . - , more serious injury to photosystem II (PSII), increase in PCD level, and autophagy activity in leaves of wheat seedlings. These results indicated that TaMCA-Id functioned in PCD through interacting with autophagy under NaCl stress, which could be used to improve the salt tolerance of crop plants.

The Calcium-Dependent Protein Kinase TaCDPK27 Positively Regulates Salt Tolerance in Wheat.

As essential calcium ion (Ca2+) sensors in plants, calcium-dependent protein kinases (CDPKs) function in regulating the environmental adaptation of plants. However, the response mechanism of CDPKs to salt stress is not well understood. In the current study, the wheat salt-responsive gene TaCDPK27 was identified. The open reading frame (ORF) of TaCDPK27 was 1875 bp, coding 624 amino acids. The predicted molecular weight and isoelectric point were 68.905 kDa and 5.6, respectively. TaCDPK27 has the closest relationship with subgroup III members of the CDPK family of rice. Increased expression of TaCDPK27 in wheat seedling roots and leaves was triggered by 150 mM NaCl treatment. TaCDPK27 was mainly located in the cytoplasm. After NaCl treatment, some of this protein was transferred to the membrane. The inhibitory effect of TaCDPK27 silencing on the growth of wheat seedlings was slight. After exposure to 150 mM NaCl for 6 days, the NaCl stress tolerance of TaCDPK27-silenced wheat seedlings was reduced, with shorter lengths of both roots and leaves compared with those of the control seedlings. Moreover, silencing of TaCDPK27 further promoted the generation of reactive oxygen species (ROS); reduced the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT); aggravated the injury to photosystem II (PS II); and increased programmed cell death (PCD) in wheat leaves under NaCl treatment, confirming that the TaCDPK27-silenced seedlings exhibited more NaCl injury than control seedlings. Taken together, the decrease in NaCl tolerance in TaCDPK27-silenced seedlings was due to excessive ROS accumulation and subsequent aggravation of the NaCl-induced PCD. TaCDPK27 may be essential for positively regulating salt tolerance in wheat seedlings.