Selective Activation of Cascade Assembly Amplification for DNA Methyltransferase Detection Using a Double-Loop Interlocked DNA Circuit.

Precise and reliable monitoring of DNA adenine methyltransferase (Dam) activity is essential for disease diagnosis and biological analysis. However, existing techniques for detecting Dam activity often rely on specific DNA recognition probes that are susceptible to DNA degradation and exhibit limited target sensitivity and specificity. In this study, we designed and engineered a stable and dynamic DNA nanodevice called the double-loop interlocked DNA circuit (DOOR) that enables the sensitive and selective monitoring of Dam activity in complex biological environments. The DOOR incorporates two interlocked specialized sequences: a palindromic sequence for Dam identification and an initiator sequence for signal amplification. In the presence of Dam, the DOOR is cleaved by double-stranded DNA phosphodiesterase I endonuclease, generating massive double-stranded DNA (dsDNA) units. These units can self-assemble into a long dsDNA scaffold, thereby enhancing the subsequent reaction kinetics. The dsDNA scaffold further triggers a hyperbranched hybrid chain reaction to produce a fluorescent 3D DNA nanonet, enabling more precise monitoring of the Dam activity. The DOOR device exhibits excellent sensitivity, specificity, and stability, rendering it a powerful tool for studying DNA methylation in various biological processes and diseases.

Nanoconfined Silver Nanoclusters Combined with X-Shaped DNA Recognizer-Triggered Cascade Amplification for Electrochemiluminescence Detection of APE1.

Apurinic/apyrimidinic endonuclease 1 (APE1), as a vital base excision repair enzyme, is essential for maintaining genomic integrity and stability, and its abnormal expression is closely associated with malignant tumors. Herein, we constructed an electrochemiluminescence (ECL) biosensor for detecting APE1 activity by combining nanoconfined ECL silver nanoclusters (Ag NCs) with X-shaped DNA recognizer-triggered cascade amplification. Specifically, the Ag NCs were prepared and confined in the glutaraldehyde-cross-linked chitosan hydrogel network using the one-pot method, resulting in a strong ECL response and exceptional stability in comparison with discrete Ag NCs. Furthermore, the self-assembled X-shaped DNA recognizers were designed for APE1 detection, which not only improved reaction kinetics due to the ordered arrangement of recognition sites but also achieved high sensitivity by utilizing the recognizer-triggered cascade amplification of strand displacement amplification (SDA) and DNAzyme catalysis. As expected, this biosensor achieved sensitive ECL detection of APE1 in the range of 1.0 × 10-3 U·µL-1 to 1.0 × 10-10 U·µL-1 with the detection limit of 2.21 × 10-11 U·µL-1, rendering it a desirable approach for biomarker detection.

Identification of unique indolylcyanoethylenyl sulfonylanilines as novel structural scaffolds of potential antibacterial agents.

The increasing incidence of antibiotic resistance has forced the development of unique antimicrobials with novel multitargeting mechanisms to combat infectious diseases caused by multidrug-resistant pathogens. Structurally unique indolylcyanoethylenyl sulfonylanilines (ISs) were exploited as novel promising antibacterial agents to confront stubborn drug resistance. Some prepared ISs possessed favorable bacteriostatic action towards the tested bacteria. Especially, hydroxyethyl IS 14a exerted 8-fold more potent inhibitory efficacy against multidrug-resistant A. baumannii and E. coli 25922 with the low MIC of 0.5 µg/mL than norfloxacin, and showed low cell toxicity and rapid bactericidal property. Moreover, this compound also possessed obvious effect of eradicating bacterial biofilm, which could effectually relieve the development of drug resistance. A preliminary assessment of the antibacterial mechanism indicated that compound 14a could disintegrate membrane integrity leading to the leakage of intracellular protein, inactivation of lactate dehydrogenase and metabolism inhibition. Hydroxyethyl IS 14a mediated the accumulation of excess reactive oxygen species, which further contributed to reducing glutathione, resulting in oxidative damage to bacteria. Furthermore, IS 14a could intercalate into DNA to hinder the biological function of DNA. Quantum chemical study disclosed that IS 14a with the lowest energy gap was conducive to displaying high bioactivity. These findings demonstrated that hydroxyethyl IS 14a as a prospective antimicrobial candidate for combating A. baumannii and E. coli 25922 would be a promising starting point.

16,17-dinor-abietane diterpenoids from Casearia kurzii.

Eleven undescribed 16,17-dinor-abietane diterpenoids, caseazins A-K (1-11), and ten known diterpenoids (12-21) were isolated from the twigs and leaves of Casearia kurzii (Flacourtiaceae). Caseazins A-K were the first abietane -type dinorditerpenoids to have been isolated from the plant of Casearia kurzii. Their chemical structures were elucidated using a combination of 1D and 2D NMR spectroscopy and mass spectrometry. The absolute configurations of 5 and 10 were established by electronic circular dichroism calculations. Moreover, compounds 2, 3, 13, 14, and 18 exhibited anti-inflammatory activity with IC50 values of 0.17, 0.36, 6.55, 1.30, and 4.53 µM, respectively. IL-1ß and caspase-1 analyses suggested that compound 14 inhibited NLRP3 inflammasome activation and blocked macrophage pyroptosis.

Target-mediated self-assembly of DNA networks for sensitive detection and intracellular imaging of APE1 in living cells.

Herein, giant DNA networks were assembled from two kinds of functionalized tetrahedral DNA nanostructures (f-TDNs) for sensitive detection and intracellular imaging of apurinic/apyrimidinic endonuclease 1 (APE1) as well as gene therapy in tumor cells. Impressively, the reaction rate of the catalytic hairpin assembly (CHA) reaction on f-TDNs was much faster than that of the conventional free CHA reaction owing to the high local concentration of hairpins, spatial confinement effect and production of giant DNA networks, which significantly enhanced the fluorescence signal to achieve sensitive detection of APE1 with a limit of 3.34 × 10-8 U µL-1. More importantly, the aptamer Sgc8 assembled on f-TDNs could enhance the targeting activity of the DNA structure to tumor cells, allowing it to endocytose into cells without any transfection reagents, which could achieve selective imaging of intracellular APE1 in living cells. Meanwhile, the siRNA carried by f-TDN1 could be accurately released to promote tumor cell apoptosis in the presence of endogenous target APE1, realizing effective and precise tumor therapy. Benefiting from the high specificity and sensitivity, the developed DNA nanostructures provide an excellent nanoplatform for precise cancer diagnosis and therapy.

Benzenesulfonyl thiazoloimines as unique multitargeting antibacterial agents towards Enterococcus faecalis.

New efficient antimicrobial agents are urgently needed to combat invasive multidrug-resistant pathogens infections. Structurally unique benzenesulfonyl thiazoloimines (BSTIs) were exploited as novel potential antibacterial victors to confront terrific drug resistance. Some developed BSTIs exerted effectively antimicrobial efficacy against the tested strains. Notably, 2-pyridyl BSTI 14d exhibited good antibacterial activity against E. faecalis with MIC value of 1 µg/mL, which was superior to sulfathiazole and norfloxacin. The most active compound 14d not only showed rapid bactericidal properties and impeded E. faecalis biofilm formation to effectually relieve the development of drug resistance, but also performed low toxicity toward human red blood cells, human normal squamous epithelial cells and human non-neoplastic colon epithelial cells. Mechanistic investigation demonstrated that molecule 14d could exert efficient membrane destruction leading to the leakage of intracellular materials and metabolism inhibition, cause oxidative damage of E. faecalis through accumulation of excess reactive oxygen species and reduction of glutathione activity, and intercalate into DNA to hinder replication of DNA. Molecular docking indicated that the formation of 14d-dihydrofolate synthetase supramolecular complex could hinder the function of this enzyme. ADME analysis displayed that compound 14d possessed promising pharmacokinetic properties. These findings suggested that the newly developed benzenesulfonyl thiazoloimines with multitargeting antibacterial potential provided a new possibility for evading resistance.

Effect of Nurse-Led Individualised Self-Care Model on Myocardial Infarction Patients with Diabetes: A Randomised Controlled Pilot Trial.

Background: To assess the effectiveness of the nurse-led individualised self-care model on myocardial infarction (MI) patients with diabetes. Methods: A total of 120 MI patients were enrolled from May 2020 to December 2021. The intervention group received the nurse-led individualised self-care model (n = 60), whereas the control group only received routine health education (n = 60). The Myocardial Infarction Dimensional Assessment Scale (MIDAS), Coronary Heart Disease Self-Management Behavior Scale (CSMS), Self-Rated Abilities for Health Practices (SRAHP) scale, General Self-Efficacy Scale (GSES), Hospital Anxiety and Depression Scale (HADS), blood glucose and nursing satisfaction in both groups were observed and recorded. Results: The six MIDAS subscales except for insecurity, and all dimensions of the CSMS, SRAHP, GSES and HADS scores, of the intervention group were significantly improved compared to those of the control group (p < 0.05). Compared with the control group (5.69 ± 1.43 mmol/L), the intervention group showed a decrease in the serum levels of fasting blood glucose (4.83 ± 1.57 mmol/L; p < 0.01). Conclusions: Our pilot study provides preliminary evidence supporting the feasibility of implementing nurse-led individualised self-care, suggesting its preliminary effects in improving health-related quality of life, self-care ability, health behaviours, self-efficacy, social support and nursing satisfaction among MI patients with diabetes. However, considering the unblinded and pilot nature of this study, these positive results should be interpreted with caution. Clinical Trial Registration: OSF Registration number: DOI 10.17605/OSF.IO/DVW95 (https://archive.org/details/osf-registrations-dvw95-v1).

Construction of Fast-Walking Tetrahedral DNA Walker with Four Arms for Sensitive Detection and Intracellular Imaging of Apurinic/Apyrimidinic Endonuclease 1.

Herein, a novel tetrahedral DNA walker with four arms was engineered to travel efficiently on the 3D-tracks via catalyzed hairpin assembly autonomously, realizing the sensitive detection and activity assessment as well as intracellular imaging of apurinic/apyrimidinic endonuclease 1 (APE1). In contrast to traditional DNA walkers, the tetrahedral DNA walker with the rigid 3D framework structure and nonplanar multi-sites walking arms endowed with high collision efficiency, showing a fast walking rate and high nuclease resistance. Impressively, the initial rate of the tetrahedral DNA walker with four arms was 4.54 times faster than that of the free bipedal DNA walker and produced a significant fluorescence recovery in about 40 min, achieving a sensitive detection of APE1 with a low detection limit of 5.54× 10-6 U/µL as well as ultrasensitive intracellular APE1 fluorescence activation imaging. This strategy provides a novel DNA walker for accurate identification of low-abundance cancer biomarker and potential medical diagnosis.

MicroRNA­181a promotes epithelial­mesenchymal transition in esophageal squamous cell carcinoma via the TGF­ß/Smad pathway.

Esophageal squamous cell carcinoma (ESCC) is one of the most debilitating and invasive tumors. Although previous reports have demonstrated the critical role microRNA­181a (miR­181a) serves in the progression of ESCC, how miR­181a induces epithelial­mesenchymal transition (EMT) remains to be elucidated. In the present study, the expression profiles of TGF­ß1 and Smad4 proteins in 88 patients with ESCC and 21 adjacent non­cancerous tissues were analyzed using immunohistochemistry. The expression of miR­181a in ESCC cells (ECA109 and TE­1) and HEEC was analyzed using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). The role of miR­181a in ESCC was analyzed using miR­181a mimics and inhibitor in the same system. Migration, proliferation and apoptosis of cells were assessed using wound­healing assays and cell proliferation assays and flow cytometry, respectively. The expression levels of TGF­ß1 and Smad4 in ESCC cell lines transfected with miR­181a mimics and inhibitor were measured using RT­qPCR and western blotting. The expression of E­cadherin and vimentin was also assessed following transfection. The findings demonstrated that expression of TGF­ß1 was upregulated, in contrast to Smad4 expression which was downregulated. Expression levels of Smad4 affected the prognosis of patients with ESCC. Higher expression of miR­181a promoted migration and proliferation but inhibited apoptosis of ESCC cells. miR­181a promoted EMT by modulating Smad4 expression in ESCC cells. Overall, these findings revealed that miR­181a induced EMT in ESCC via the TGF­ß/Smad pathway in ESCC. Consequently, miR­181a is a potential novel target against ESCC.

Autophagy-mediated compartmental cytoplasmic deletion is essential for tobacco pollen germination and male fertility.

In plants, macroautophagy/autophagy has mainly been associated with stress-related processes but how it impacts normal physiological and developmental processes remains largely unexplored. Pollen germination is the critical first step toward fertilization in flowering plants. It is metabolically demanding and relies on high levels of cytoplasmic reorganization activities to support a dramatic morphological transformation that underlies the development of a pollen tube as the conduit to deliver sperm for fertilization. The role of autophagy in this process remains unclear. Here we provide evidence that pollen germination is accompanied by elevated autophagic activity and successful pollen tube emergence depends on autophagy-mediated cytoplasmic deletion. Genetic and cytological experiments demonstrate that inhibition of autophagy prevents pollen germination while induces the persistence of a layer of undegraded cytoplasm at the germination aperture. Together, these results unveil a novel compartmentalized autophagy. Furthermore, high-throughput comparative lipidomic analyses show that suppressed autophagy-induced inhibition of pollen germination is accompanied by altered profiles of stored and signaling lipids. Proteomic analyses reveal that autophagy likely exert its role in pollen germination via downstream mitochondria-related pathways. These findings reveal a critical role for autophagy in initiating pollen germination and provide evidences for compartmental cytoplasmic deletion being crucial for male fertility. Abbreviations: 3-MA: 3-methyladenine; ATG: autophagy-related gene; Cer: ceramide; CL: cardiolipin; Con A: concanamycin A; DAG: diradylglycerol; GO: gene ontology; HAG: hour after germination; LC-MS: liquid chromatography-mass spectrometry; MAG: min after germination; MDC: monodansylcadaverine; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PLD: phospholipase D; PtdIns3K: phosphatidylinositol 3-kinase; RT-qPCR: quantitative real-time reverse transcription PCR; TAG: triradylglycerol; TEM: transmission electron microscopy; TMT: tandem mass tagging.

Sex-differential effects of olanzapine vs. aripiprazole on glucose and lipid metabolism in first-episode schizophrenia

Abstract Objective To compare sex difference in metabolic effect of olanzapine versus aripiprazole on schizophrenia. Methods A twelve-week prospective open-label cohort study to compare four subgroups according to first-episode schizophrenia patients' type of drug usage and sex: female aripiprazole (n = 11), male aripiprazole (n = 11), female olanzapine (n = 10), and male olanzapine (n = 11) for body mass index, fasting serum triglyceride, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and fasting glucose. Results Aripiprazole may be associated with weight gain in female patients with low-baseline weight. Aripiprazole may have an adverse effect of weight and favorable effects of circulating glucose and lipid on female over male schizophrenia patients. The aripiprazole-induced changes in glucose and lipid may be independent of body fat storage, especially for female schizophrenia patients. Olanzapine may have adverse effects of weight, glucose and lipid profiles on female over male schizophrenic patients. Discussion Our findings fill the gap in knowledge and provide a sex-specific guidance to psychiatrist better tailoring treatment to individual sex-differential characteristics and a key clue to understand the sex-differential mechanism of antipsychotics-induced metabolic dysfunction.

Papain-like and legumain-like proteases in rice: genome-wide identification, comprehensive gene feature characterization and expression analysis.

BACKGROUND: Papain-like and legumain-like proteases are proteolytic enzymes which play key roles in plant development, senescence and defense. The activities of proteases in both families could be inhibited by a group of small proteins called cystatin. Cystatin family genes have been well characterized both in tobacco and rice, suggesting their potential roles in seed development. However, their potential targets, papain-like and legumain-like proteases, have not been well characterized in plants, especially in rice, a model plant for cereal biology. RESULTS: Here, 33 papain-like and 5 legumain-like proteases have been identified in rice genome, respectively. Gene structure, distribution in rice chromosome, and evolutionary relationship to their counterparts in other plants have been well characterized. Comprehensive expression profile analysis revealed that two family genes display divergent expression pattern, which are regulated temporally and spatially during the process of seed development and germination. Our experiments also revealed that the expression of most genes in these two families is sensitively responsive to plant hormones and different abiotic stresses. CONCLUSIONS: Genome-wide identification and comprehensive gene expression pattern analysis of papain-like and legumain-like proteases in rice suggests their multiple and cooperative roles in seed development and response to environmental variations, which provides several useful cues for further in-depth study.

Effects of zearalenone on the localization and expression of the growth hormone receptor gene in the uteri of post-weaning piglets.

OBJECTIVE: In this study, we investigated the adverse effects of dietary zearalenone (ZEA) (0.5 to 1.5 mg/kg diet) on the localization and expression of the growth hormone receptor (GHR) in the uteri of post-weaning gilts and explored alternative mechanism of the reproductive toxicity of ZEA on piglets. METHODS: A total of forty healthy piglets (Duroc×Landrace×Large White) aged 28 d were selected for study. Piglets were transferred to single cages after 10 days' adaptation on an obstetric table. The animals were allocated to one of four treatments: a normal basal diet supplemented with 0 (Control), 0.5 (ZEA0.5), 1.0 (ZEA1.0), or 1.5 (ZEA1.5) mg/kg purified ZEA, and fed for 35 d after the 10-d adaptation. Analyzed ZEA concentrations in the diets were 0, 0.52±0.07, 1.04±0.03, and 1.51±0.13 mg/kg, respectively. At the end of the feeding trial, piglets were euthanized after being fasted for 12 h. Two samples of uterine tissue from each pig were rapidly collected, one of which was stored at -80°C for analysis of the relative mRNA and protein expression of GHR, and the second was promptly fixed in Bouin's solution for immunohistochemical analysis. RESULTS: The relative weight of the uteri and thickness of the myometrium and endometrium increased linearly (p<0.001) and quadratically (p<0.001) with an increasing level of ZEA. The results of immunohistochemical analysis indicated that GHR immunoreactive substance was mainly localizated in the cytoplasm of uterine smooth muscle, glandular epithelial, luminal epithelial, stromal, and vascular endothelial cells. In contrast, nuclear staining was rarely observed. The immunoreactive integrated optic density of GHR in the myometrium, luminal epithelium, glandular epithelium, and whole uteri of weaning gilts increased linearly (p<0.001) and quadratically (p<0.05) with an increasing level of ZEA. The mRNA and protein expression of GHR in the uteri of weaning gilts increased linearly (p<0.001) and quadratically (p<0.05) with an increasing level of ZEA. CONCLUSION: In conclusion, ZEA at a concentration of 0.5 mg/kg was sufficient to significantly thicken the myometrium and endometrium, and at a concentration of 1.0 mg/kg induced a high level of GHR expression to promote growth and development of the uteri. This revealed an alternative molecular mechanism whereby ZEA induces growth and development of the uteri and provides a theoretical basis for the revision of Chinese feed hygiene standards.

Autophagy in sexual plant reproduction as an emerging field.

Autophagy is an evolutionary conserved system for clearing and recycling cellular components. Critical roles of autophagy in the responses of plant to different environmental stresses have been revealed during past decades. However, little is known about the role of autophagy in plant developmental processes, especially in the processes of sexual plant reproduction. Here, we briefly discuss recent advances in the emerging field and wish to bring some potential research directions into attention. Possible divergence of molecular mechanism of autophagy in respect to the current debatable view is also discussed.

[Risk Assessment of Malaria in Libo County, Guizhou Province].

Data concerning malaria endemic situation during 2006-2012 and data concerning malaria transmission risk and malaria elimination capacity during 2010-2012 were collected. The results showed that during 2006-2012, malaria in Libo County was predominated by vivax malaria with the transmission vector of Anopheles sinensis, and malaria incidence declined year by year（3.72/10 000, 3.56/10 000, 5.76/10 000, 4.34/10 000, 2.54/10 000, 1.14/10 000, and 0, respectively）. In the residents surveyed during 2010-2012, >2% received blood test, 82.9%（29/35） received standard therapy, 93.3%（651/698）had usage of insect-resistant facilities, and 440 received medical training, with an awareness rate of 92.3%（738/800）in the residents. The malaria transmission risk index of Anopheles mosquito was 2, the area risk value was 10, and the malaria transmission risk index was 20, indicating a moderately-low level of risk.

[Measures and effect of malaria prevention and control in Libo County].

OBJECTIVE: To understand the implementation status and effect of prevention and control of malaria in Libo County, so as to provide the evidence for improving the malaria elimination working. METHODS: The data about malaria from the county CDC and county hospital were collected and 16 villages from 8 townships were randomly sampled and 10 villagers of each village were investigated. Other information about the prevention and control of malaria was also investigated. RESULTS: The incidence of malaria was decreasing annually, from 5.75 per 10,000 in 2008 to zero in 2012. The malaria monitoring could be well conducted in the county and township levels. The infection source could be controlled in time. The utilization rate of anti-mosquito facilities in the residents was 93.25% and the awareness rate of knowledge about malaria prevention and control was 40.13%. CONCLUSIONS: The implementation and effect of prevention and control of malaria are satisfactory in Libo County, but the medium control is limited and the active protection consciousness of the residents is not strong. Therefore, the task of malaria elimination is still very arduous.

A comprehensive, genome-wide analysis of autophagy-related genes identified in tobacco suggests a central role of autophagy in plant response to various environmental cues.

Autophagy is an evolutionarily conserved mechanism in both animals and plants, which has been shown to be involved in various essential developmental processes in plants. Nicotiana tabacum is considered to be an ideal model plant and has been widely used for the study of the roles of autophagy in the processes of plant development and in the response to various stresses. However, only a few autophagy-related genes (ATGs) have been identified in tobacco up to now. Here, we identified 30 ATGs belonging to 16 different groups in tobacco through a genome-wide survey. Comprehensive expression profile analysis reveals an abroad expression pattern of these ATGs, which could be detected in all tissues tested under normal growth conditions. Our series tests further reveal that majority of ATGs are sensitive and responsive to different stresses including nutrient starvation, plant hormones, heavy metal and other abiotic stresses, suggesting a central role of autophagy, likely as an effector, in plant response to various environmental cues. This work offers a detailed survey of all ATGs in tobacco and also suggests manifold functions of autophagy in both normal plant growth and plant response to environmental stresses.

Genome-wide identification and characterization of cystatin family genes in rice (Oryza sativa L.).

KEY MESSEAGE: 11 Cystatin genes in rice were identified, and their expression patterns were comprehensively analyzed, which reveals multiple roles in both seed development and plant response to environmental variations. Cystatin is a group of small proteins and known to inhibit the activities of cysteine proteases in the papain C1A and legumain C13 peptidase families in plants. Cystatin family genes have only been well characterized recently in a few plant species such as Hordeum vulgare and Nicotiana tabacum, which show their critical roles in programmed cell death and responses to biotic stresses. Up to now, little is known about cystatin family genes and their roles in Oryza sativa, a model plant for cereal biology study. Here, we identified 11 cystatin genes in rice genome. Comprehensive expression profile analysis reveals that cystatin family genes in rice display diverse expression pattern. They are temporally regulated at different developmental stages during the process of seed production and germination. Our experiments also reveal that the majority of cystatin genes are responsive to plant hormones and different environmental cues including cold, drought and other abiotic stresses, while some others are very stable under different stresses, indicating their fundamental roles in normal plant development. In addition, their distribution in rice chromosomes and their evolutionary relation to the members of Cystatin family in A. thaliana and N. tabacum have also been analyzed. These works suggest multiple roles of cystatin family genes in both seed development and plant response to environmental variations.

Comprehensive analysis of cystatin family genes suggests their putative functions in sexual reproduction, embryogenesis, and seed formation.

Cystatins are tightly bound and reversible inhibitors of cysteine proteases in C1A and C13 peptidase families, which have been identified in several species and shown to function in vegetative development and response to biotic/abiotic stresses in plants. Recent work revealed their critical role in regulating programmed cell death during embryogenesis in tobacco and suggested their more comprehensive roles in the process of sexual plant reproduction, although little is known about cystatin family genes in the processes. Here, 10 cystatin family genes in Nicotiana tabacum were identified using an expressed sequence tag (EST)-based gene clone strategy. Analysis of their biochemical properties showed that nine of them have the potency to inhibit the activities of both commercial cathepsin L-like proteases and extracted cysteine proteases from seeds, but with different K i values depending on the types of proteases and the developmental stages of the seed tested. This suggests that cystatin-dependent cathepsin L-like proteolytic pathways are probably important for early seed development. Comprehensive expression profile analysis revealed that cystatin family genes showed manifold variations in their transcription levels in different plant cell types, including the sperm, egg, and zygote, especially in the embryo and seed at different developmental stages. More interestingly, intracellular localization analysis of each cystatin revealed that most members of cystatin families are recognized as secretory proteins with signal peptides that direct them to the endoplasmic reticulum. These results suggest their widespread roles in cell fate determination and cell-cell communication in the process of sexual reproduction, especially in gamete and embryo development, as well as in seed formation.

A bipartite molecular module controls cell death activation in the Basal cell lineage of plant embryos.

Plant zygote divides asymmetrically into an apical cell that develops into the embryo proper and a basal cell that generates the suspensor, a vital organ functioning as a conduit of nutrients and growth factors to the embryo proper. After the suspensor has fulfilled its function, it is removed by programmed cell death (PCD) at the late stages of embryogenesis. The molecular trigger of this PCD is unknown. Here we use tobacco (Nicotiana tabacum) embryogenesis as a model system to demonstrate that the mechanism triggering suspensor PCD is based on the antagonistic action of two proteins: a protease inhibitor, cystatin NtCYS, and its target, cathepsin H-like protease NtCP14. NtCYS is expressed in the basal cell of the proembryo, where encoded cystatin binds to and inhibits NtCP14, thereby preventing precocious onset of PCD. The anti-cell death effect of NtCYS is transcriptionally regulated and is repressed at the 32-celled embryo stage, leading to increased NtCP14 activity and initiation of PCD. Silencing of NtCYS or overexpression of NtCP14 induces precocious cell death in the basal cell lineage causing embryonic arrest and seed abortion. Conversely, overexpression of NtCYS or silencing of NtCP14 leads to profound delay of suspensor PCD. Our results demonstrate that NtCYS-mediated inhibition of NtCP14 protease acts as a bipartite molecular module to control initiation of PCD in the basal cell lineage of plant embryos.