Transcriptomic and metabolomic dissection of skeletal muscle of crossbred Chongming white goats with different meat production performance.

BACKGROUND: The transcriptome and metabolome dissection of the skeletal muscle of high- and low- growing individuals from a crossbred population of the indigenous Chongming white goat and the Boer goat were performed to discover the potential functional differentially expressed genes (DEGs) and differential expression metabolites (DEMs). RESULTS: A total of 2812 DEGs were detected in 6 groups at three time stages (3,6,12 Month) in skeletal muscle using the RNA-seq method. A DEGs set containing seven muscle function related genes (TNNT1, TNNC1, TNNI1, MYBPC2, MYL2, MHY7, and CSRP3) was discovered, and their expression tended to increase as goat muscle development progressed. Seven DEGs (TNNT1, FABP3, TPM3, DES, PPP1R27, RCAN1, LMOD2) in the skeletal muscle of goats in the fast-growing and slow-growing groups was verified their expression difference by reverse transcription-quantitative polymerase chain reaction. Further, through the Liquid chromatography-mass spectrometry (LC-MS) approach, a total of 183 DEMs in various groups of the muscle samples and these DEMs such as Queuine and Keto-PGF1α, which demonstrated different abundance between the goat fast-growing group and slow-growing group. Through weighted correlation network analysis (WGCNA), the study correlated the DEGs with the DEMs and identified 4 DEGs modules associated with 18 metabolites. CONCLUSION: This study benefits to dissection candidate genes and regulatory networks related to goat meat production performance, and the joint analysis of transcriptomic and metabolomic data provided insights into the study of goat muscle development.

Enhancing cryopreservation survival in Petunia × Calibrachoa 'Light Yellow' callus: Insights into material characteristics and genetic integrity.

Petunia × Calibrachoa 'Light Yellow' (× Petchoa 'Light Yellow') is a kind of perennial herbaceous flower obtained through intergeneric hybridization of Petunia and Calibrachoa with high ornamental value and wide application, facing challenges in seed acquisition. Expanding propagation through tissue culture is an economically efficient means. Hence, establishing an effective procedure for the storage of callus is essential for × Petchoa 'Light Yellow'. Cryopreservation is an effective method for the in vitro propagation and long-term preservation of × Petchoa 'Light Yellow' germplasms. For formulating the optimization of the vitrification procedure, first, an orthogonal experimental design was employed to pinpoint critical steps in the vitrification protocol (pre-culture, osmoprotection, dehydration, and dilution) for Petunia × Calibrachoa callus tissues and then five additional factors (pre-culture, osmoprotection I and II, dehydration, and dilution) were optimized to further reduce the sample water content and enhance cell viability levels. The vitrification procedure was described as follows: callus tissues were precultured in MS solid medium with 0.3 M sucrose for 5 d, incubated with osmoprotection solution I and II for 15 min at 25 °C, respectively, cryoprotected with PVS2 for 30 min at 0 °C, and rapidly immersed in liquid nitrogen. Cryopreserved callus tissues were then diluted in MS liquid medium with 1.2 M sucrose for 20 min at 25 °C and recovered on MS solid medium with 0.5 mg/L 6-BA and 0.1 mg/L NAA, and sucrose. The cell viability measured by TTC staining was approximately 16 %-18 % after 72 h-recovery. Following 45 days, the relative survival of callus reached up to 49.48 %. Furthermore, EST-SSR analysis showed no significant difference in the genetic stability of cryopreserved callus compared to the control. Based on the cryopreservation of × Petchoa 'Light Yellow' callus, we further evaluated the response of callus water contents to the osmotic stress in the optimized and original protocols (CK) for a higher cryopreservation survival. A comparative analysis of water content demonstrated that the procedure of gradual and gentle dehydration significantly improved water content and cell survival. Ultrastructural changes between cryopreserved and non-cryopreserved callus were examined and high vacuolation emerged as a key determinant, indicating its substantial impact on the low survival of cryopreserved cells, which should help us to understand the effectiveness of osmotic protectants in dehydration.

Iron accumulation and axonal damage of cerebellum in idiopathic cervical dystonia.

BACKGROUND AND PURPOSE: Postmortem brain study indicated that cerebellar Purkinje cell (PC) loss might be a pathological finding in patients with inherited and idiopathic cervical dystonia (ICD). The analysis of conventional magnetic resonance imaging brain scans failed to yield support for this finding. Previous studies have identified that iron overload can be the consequence of neuron death. The objectives of this study were to investigate iron distribution and demonstrate changes in axons in the cerebellum, providing evidence for PC loss in patients with ICD. METHODS: Twenty-eight patients with ICD (20 females) and 28 age- and sex-matched healthy controls were recruited. A spatially unbiased infratentorial template was applied to perform cerebellum optimized quantitative susceptibility mapping and diffusion tensor analysis based on magnetic resonance imaging. Voxel-wise analysis was performed to assess cerebellar tissue magnetic susceptibility and fractional anisotropy (FA) alterations, and the clinical relevance of these findings was investigated in the patients with ICD. RESULTS: Increased susceptibility values revealed by quantitative susceptibility mapping in the right lobule CrusI, CrusII, VIIb, VIIIa, VIIIb and IX were found in the patients with ICD. A reduced FA value was found across almost all the cerebellum; an FA value of the significant clusters within the right lobule VIIIa significantly correlated with the motor severity of patients with ICD (r = -0.575, p = 0.002). CONCLUSIONS: Our study provided evidence for cerebellar iron overload and axonal damage in patients with ICD, which may indicate PC loss and related axonal changes. These results provide evidence for the neuropathological findings in patients with ICD and further highlight the cerebellar involvement in the pathophysiology of dystonia.

CCL7 and TGF-ß secreted by MSCs play opposite roles in regulating CRC metastasis in a KLF5/CXCL5-dependent manner.

CXCL5 is overexpressed in colorectal cancer (CRC) and promotes distant metastasis and angiogenesis of tumors; however, the underlying mechanism that mediates CXCL5 overexpression in CRC remains unclear. Here, we successfully extracted and identified primary mesenchymal stromal cells (MSCs) and verified the promoting effects of tumor-associated MSCs on CRC proliferation and metastasis in vivo and in vitro. We found that MSCs not only promoted the expression of CXCL5 by secreting CCL7 but also secreted TGF-ß to inhibit this process. After secretion, CCL7/CCR1 activated downstream CBP/P300 to acetylate KLF5 to promote CXCL5 transcription, while TGF-ß reversed the effect of KLF5 on transcription activation by regulating SMAD4. Taken together, our results indicate that MSCs in the tumor microenvironment promoted the progression and metastasis of CRC and regulated the expression of CXCL5 in CRC cells by secreting CCL7 and TGF-ß. KLF5 is the key site of these processes and plays a dual role in CXCL5 regulation. MSCs and their secreted factors may serve as potential therapeutic targets in the tumor environment.

Targeted single-cell RNA sequencing analysis reveals metabolic reprogramming and the ferroptosis-resistant state in hematologic malignancies.

Hematologic malignancies are the most common hematopoietic diseases and a major public health concern. However, the mechanisms underlying myeloid tumors remain unknown owing to the intricate interplay between mutations and diverse clonal evolution patterns, as evidenced by the analysis of bulk cell-derived omics data. Several single-cell omics techniques have been used to characterize the hierarchies and altered immune microenvironments of hematologic malignancies. The comprehensive single-cell atlas of hematologic malignancies provides novel opportunities for personalized combinatorial targeted treatments, avoiding unwanted chemo-toxicity. In the present study, we performed transcriptome sequencing by combining single-cell RNA sequencing (scRNA-seq) with a targeted oncogenic gene panel for acute myeloid leukemia, overcoming the limitations of scRNA-seq in detecting oncogenic mutations. The distribution of oncogenic IDH1, IDH2, and KRAS mutations in each cell type was identified in the bone marrow (BM) samples of each patient. Our findings suggest that ferroptosis and metabolic reprogramming are involved in the tumorigenesis and chemotherapy resistance of oncogenic mutation-carrying cells. Biological progression via IDH1, IDH2, and KRAS mutations arrests hematopoietic maturation. Our study findings provide a rationale for using primary BM cells for personalized treatment in clinical settings.

IRAPs in Combination with Highly Informative ISSRs Confer Effective Potentials for Genetic Diversity and Fidelity Assessment in Rhododendron.

The species belonging to the Rhododendron genus are well-known for their colorful corolla. Molecular marker systems have the potential to elucidate genetic diversity as well as to assess genetic fidelity in rhododendrons. In the present study, the reverse transcription domains of long terminal repeat retrotransposons were cloned from rhododendrons and used to develop an inter-retrotransposon amplified polymorphism (IRAP) marker system. Subsequently, 198 polymorphic loci were generated from the IRAP and inter-simple sequence repeat (ISSR) markers, of which 119 were derived from the IRAP markers. It was shown that in rhododendrons, IRAP markers were superior to the ISSRs in some polymorphic parameters, such as the average number of polymorphic loci (14.88 versus 13.17). The combination of the IRAP and ISSR systems was more discriminative for detecting 46 rhododendron accessions than each of the systems on their own. Furthermore, IRAP markers demonstrated more efficiency in genetic fidelity detection of in-vitro-grown R. bailiense Y.P.Ma, C.Q.Zhang and D.F.Chamb, an endangered species recently recorded in Guizhzhou Province, China. The available evidence revealed the distinct properties of IRAP and ISSR markers in the rhododendron-associated applications, and highlighted the availability of highly informative ISSR and IRAP markers in the evaluation of genetic diversity and genetic fidelity of rhododendrons, which may facilitate preservation and genetic breeding of rhododendron plants.

Correlating the Solvating Power of Solvents with the Strength of Ion-Dipole Interaction in Electrolytes of Lithium-ion Batteries.

The solvation structure of Li+ plays a significant role in determining the physicochemical properties of electrolytes. However, to date, there is still no clear definition of the solvating power of different electrolyte solvents, and even the solvents that preferentially participate in the solvation structure remain controversial. In this study, we comprehensively discuss the solvating power and solvation process of Li+ ions using both experimental characterizations and theoretical calculations. Our findings reveal that the solvating power is dependent on the strength of the Li+ -solvent (ion-dipole) interaction. Additionally, we uncover that the anions tend to enter the solvation sheath in most electrolyte systems through Li+ -anion (ion-ion) interaction, which is weakened by the shielding effect of solvents. The competition between the Li+ -solvent and Li+ -anion interactions ultimately determines the final solvation structures. This insight into the fundamental understanding of the solvation structure of Li+ provides inspiration for the design of multifunctional mixed-solvent electrolytes for advanced batteries.

Inactivated STAT5 pathway underlies a novel inhibitory role of EBF1 in chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) is a disease caused by gradual accumulation of functionally incompetent lymphocytes. The majority of CLL cases are accompanied by chemoresistance. Early B cell factor 1 (EBF1) is a crucial contributor to B-cell lymphopoiesis. This study is to explore the effect of EBF1 on CLL cell progression and its involvement in regulating the signal transducers and activators of transcription 5 (STAT5) pathway. We conducted a correlation analysis between EBF1 and the clinical characteristics of CLL patients. Subsequently, EBF1 was overexpressed by transfection with EBF1 overexpression plasmid and the STAT5 pathway was also blocked by treatment with SH-4-54 in isolated CD20+ B lymphocytes to investigate their roles in the regulation of cellular functions. STAT5, Janus kinase 2 (JAK2) expression and their phosphorylation levels were determined by quantitative PCR and Western blot analyses. The in vivo effects of EBF1 on tumor growth were evaluated using a xenotransplant model. Downregulation of EBF1 was observed in CD20+ B lymphocytes of CLL patients. EBF1 overexpression disrupted the activation of STAT5 pathway, as evidenced by decreased expression and phosphorylation levels of STAT5 and JAK2. Furthermore, overexpression of EBF1 repressed viability and cell cycle entry, and increased apoptosis of CD20+ B lymphocytes by inhibiting the STAT5 pathway. Finally, EBF1 exerted antitumor effects in nude mice. Overall, our study elucidates the inhibitory role of EBF1 in CLL through inactivation of the STAT5 pathway, which may provide new targets for CLL treatment.

Strongylocentrotus nudos Egg Polysaccharide induces autophagy and apoptosis in leukaemia cells by regulating mitochondrial function.

In this study, we investigated the ability of the Polysaccharide from the Eggs of Strongylocentrotus nudus (SEP) to regulate cellular autophagy and apoptosis in leukaemia cells. Human acute myeloid leukaemia (AML) cells (HL60) and murine AML cells (L1210) treated with SEP were used to assess viability using Cell Counting Kit-8, cytotoxicity by measuring lactate dehydrogenase release, the generation of reactive oxygen species (ROS) by DCFH-DA staining. In addition, we utilized a mouse model of leukaemia in which L1210 cells were injected into DBA/2 mice by sub-axillary injection. Treatment with SEP decreased cell viability, increased in cytotoxicity and increased the release of ROS in a dose-dependent manner. SEP treatment was also associated with the activation of pro-apoptotic proteins cleaved caspase-3, cleaved caspase-9 and cleaved poly (ADP-ribose) polymerase (PARP). Activation of the apoptotic pathway led to the release of cytochrome C (CytoC) into the cytosol of the cell resulting in decreased membrane potential. The effect of SEP treatment was depended on the activation of the nuclear factor kappa-B (NF-κB) signalling pathway as SEP treatment led to an increase in NF-κB phosphorylation, and inhibition of NF-κB signalling using PDTC blocked SEP-mediated activation of apoptosis. Treatment with SEP also prolonged survival time in our leukaemia mouse model and was associated with diminished tumour volume, increased leucocyte and lymphocyte proliferation, promoted pro-inflammatory factor release in serum and enhanced immune function. Taken together, these data suggest that SEP inhibits the progression of leukaemia by initiating mitochondrial dysfunction, autophagy, and apoptosis via the NF-κB signalling pathway.

ApSerpin-ZX from Agapanthus praecox, is a potential cryoprotective agent to plant cryopreservation.

Cryopreservation-induced cell death is regarded as an important problem faced by cryobiologists. Oxidative stress and programmed cell death are detrimental to cell survival. Serine protease inhibitors (serpins) inhibit pro-cell-death proteases and play a pro-survival role in excessive cell death induced by abiotic stress. In this study, ApSerpin-ZX was isolated from Agapanthus praecox and characterized as a protective protein in plant cryopreservation. The mRNA level of ApSerpin-ZX was elevated under abiotic stress, such as salt, osmosis, oxidative, cold, and cryoinjury. The purified recombinant protein expressed in E. coli was added to the plant vitrification solution and used for A. praecox embryogenic callus cryopreservation. The concentration of 0.6-4.8 mg∙L-1 of ApSerpin-ZX protein was beneficial to the survival of cryopreserved embryogenic callus of A. praecox. The most effective concentration was 1.2 mg∙L-1, which elevated the survival by 37.15%. Subsequently, the cryopreservation procedure with 1.2 mg∙L-1 of ApSerpin-ZX protein was regarded as the treated group, compared to standard procedure, to determine the physiological mechanism of ApSerpin-ZX protein on cryopreserved cell. The MDA and H2O2 contents were significantly decreased in the treated group, along with reduced OH· generation activity in the recovery stage. After the addition of ApSerpin-ZX, the POD and CAT activities keep increased, while SOD activity increased only after dehydration. Besides, the caspase-1-like and caspase-3-like activities were lower than the standard procedure. This study indicated that ApSerpin-ZX was a potential cryoprotective agent that alleviated oxidative stress and cell death induced by cryopreservation.

Development and neutralization analysis of recombinant BVDVs expressing a CSF single chain antibody in vitro.

Although the immunization against swine fever (SF) is compulsory in China, it has still emerged in several areas at times. Herein, this study was conducted to develop an antibody vaccine which can clear the classical swine fever virus (CSFV) immediately after the pathogen invasion. Bovine viral diarrhoea virus (BVDV) infectious cDNA clone pASH28 was used to express a single-chain fragment variable (scFv) antibody against CSFV (CSFV/scFv) by reverse genetic technique. CSFV/scFv was inserted at the N-terminus of the C or Erns gene, generating two rBVDVs (rBVDV/C-CSFV/scFv and rBVDV/Erns-CSFV/scFv). Although both the rBVDVs could stably propagate on MDBK cells, different cellular characteristics existed. Obvious green fluorescence against the CSFV/scFv antibody could be visual on the cytomembrane or outside of the cells infected with rBVDV/Erns-CSFV/scFv, while much weaker fluorescence was observed in rBVDV/C-CSFV/scFv - infected cells. The CSFV/scFv antibodies induced by the two rBVDVs could recognize CSFV, but the rBVDV/Erns-CSFV/scFv induced stronger viral neutralization reaction. It was speculated that the neutralization activity might be associated with the expression location of CSFV/scFv antibody. The datas in this study provide evidence that rBVDV/Erns-CSFV/scFv may be engineered as a new antibody vaccine candidate against CSFV in the future.

Downregulated PITX1 Modulated by MiR-19a-3p Promotes Cell Malignancy and Predicts a Poor Prognosis of Gastric Cancer by Affecting Transcriptionally Activated PDCD5.

BACKGROUND/AIMS: PITX1 has been identified as a potential tumor-suppressor gene in several malignant tumors. The molecular mechanism underlying PITX1, particularly its function as a transcription factor regulating gene expression during tumorigenesis, is still poorly understood. METHODS: The expression level and location of PITX1 were determined by quantitative reverse transcription PCR (qRT-PCR) and immunohistochemical staining in gastric cancer (GC). The effect of PITX1 on the GC cell proliferation and tumorigenesis was analyzed in vitro and in vivo. To explore how PITX1 suppresses cell proliferation, we used PITX1-ChIP-sequencing to measure genome-wide binding sites of PITX1 and assessed global function associations based on its putative target genes. ChIP-PCR, electrophoretic mobility shift assay, and promoter reporter assays examined whether PITX1 bound to PDCD5 and regulated its expression. The function of PDCD5 in GC cell apoptosis was further examined in vitro and in vivo. The relationship between the PITX1 protein level and GC patient prognosis was evaluated by the Kaplan-Meier estimator. Meanwhile, the expression level of miR-19a-3p, which is related to PITX1, was also detected by luciferase reporter assay, qRT-PCR, and western blotting. RESULTS: The expression level of PITX1 was decreased in GC tissues and cell lines. Elevated PITX1 expression significantly suppressed the cell proliferation of GC cells and tumorigenesis in vitro and in vivo. PITX1 knockdown blocked its inhibition of GC cell proliferation. PITX1 bound to whole genome-wide sites, with these targets enriched on genes with functions mainly related to cell growth and apoptosis. PITX1 bound to PDCD5, an apoptosis-related gene, during tumorigenesis, and cis-regulated PDCD5 expression. Increased PDCD5 expression in GC cells not only induced GC cell apoptosis, but also suppressed GC cell growth in vitro and in vivo. Moreover, PITX1 expression was regulated by miR-19a-3p. More importantly, a decreased level of PITX1 protein was correlated with poor GC patient prognosis. CONCLUSION: Decreased expression of PITX1 predicts shorter overall survival in GC patients. As a transcriptional activator, PITX1 regulates apoptosis-related genes, including PDCD5, during gastric carcinogenesis. These data indicate PDCD5 to be a novel and feasible therapeutic target for GC.

Cloning and characterization of ApCystatin, a plant cystatin gene from Agapanthus praecox ssp. orientalis responds to abiotic stress.

Plant cystatins are involved in the regulation of protein turnover and play important roles in defense mechanisms. We cloned the ApCystatin gene from Agapanthus praecox ssp. orientalis, a famous ornamental and medical plant. The complete cDNA sequence of ApCystatin is comprised of 1439 nucleotides with a 423 bp ORF encoding 140 amino acids. The mRNA level of ApCystatin was significantly up-regulated under various abiotic stress, such as salt, osmosis, oxidative and cold stresses, which suggested that ApCystatin participated in the plant's resistance to stress. The recombinant ApCystatin fusion protein expressed in E. coli transetta (DE3) cells was approximate 18 kDa. 25 µg of ApCystatin inhibited more than 95% activity of papain, suggesting ApCystatin as a papain-like protease inhibitor. As an exogenous substance, 1.60 µg/mL ApCystatin protein improved the regrowth percentage of Arabidopsis 60-h seedlings after cryopreservation from 30% to 47%. In addition, the relative survival rate of A. praecox embryogenic callus after cryopreservation also increased for 30% with addition of 1.20 µg/mL ApCystatin protein. This indicated that ApCystatin performed protective property against cryoinjury to Arabidopsis 60-h seedlings and A. praecox embryogenic callus during cryopreservation. Under various abiotic stress conditions, the recombinant ApCystatin protein showed significant advantage in growth rates at NaCl, mannitol, PEG6000, cold, acidic and alkaline conditions, compared to control. In conclusion, ApCystatin as a new member of plant cystatins exhibited protective property against cryoinjury in plant cryopreservation and abiotic stress in E. coli.

Preoperative diagnosis and prognosis in 40 Parathyroid Carcinoma Patients.

OBJECTIVE: Parathyroid carcinoma (PC) is a rare disease which is difficult to diagnose preoperatively and predict prognosis. The goal of this study was to analyse the preoperative predictive factors and prognostic factors in PC patients and to evaluate the possibility of diagnosing PC preoperatively. DESIGN, SETTING AND PATIENTS: This is a retrospective study from Jan 2000 to Aug 2015 conducted in Shanghai Ruijin Hospital. MEASUREMENTS: Comparisons were made between 40 parathyroid carcinoma patients and 282 patients with benign parathyroid lesions during the same period. All patients underwent parathyroid surgery, and the results were certified by paraffin pathology. Prognostic factors were analysed in the 40 PC patients. RESULTS: Patients with higher levels of intact parathyroid hormone (P < 0·001, OR = 1·001, CI: 1·000-1·002), calcium (P = 0·008, OR = 3·395, CI: 1·382-8·341) and a larger parathyroid volume (P = 0·001, OR = 2·023, CI: 1·333-3·071) were more likely to have PC. Local excision (P = 0·008, OR = 4·992, CI: 1·533-16·252), stage III in the Schulte staging system (P = 0·039, OR = 9·600, CI: 1·12-82·322), high risk in the Schulte Risk Classification (P = 0·012, OR = 5·466, CI: 1·448-20·628) and first surgery by other medical teams (P = 0·008, OR = 4·992, CI: 1·496-15·037) were associated with PC recurrence. Calcium (P = 0·01, OR = 7·270, CI: 1·611-32·812), intact parathyroid hormone (P = 0·037, OR = 1·001, CI: 1·000-1·001), local excision (P = 0·009, OR = 6·875, CI: 1·633-28·936) and recurrence (P = 0·014, OR = 7·762, CI: 1·504-40·055) were associated with death. CONCLUSIONS: A preoperative diagnostic system may provide a new method to distinguish PC from benign parathyroid lesions before surgery. For PC patients who did not undergo en-bloc resection at first operation, timely further surgery may offer a second chance of cure. Early diagnosis and surgery are pivotal to reduce mortality in PC patients.

Prognostic utility of combination of NT-proBNP with high sensitive cTn I in patients with heart failure: Results from retrospective study in an emergency department.

BACKGROUND: N-terminal proBNP (NT-proBNP) and cardiac troponin I (cTn I) are widely used for the diagnosis of myocardial injury, but have not been used for routine evaluation in heart failure (HF) population. AIMS: To evaluate the prognostic utility of combination of NT-proBNP and cTn I in patients with HF, including serial NT-proBNP/cTn I measurements and discharge NT-proBNP/cTn I levels. PATIENTS AND METHODS: A total of 610 patients presenting in our emergency department for acute HF were studied. The mortality and HF-related readmission were endpoints in the study. NT-proBNP and cTn I were tested on admission including first 5 consecutive days, and on discharge. RESULTS: A discharge cTn I cut-off value at 24 ng/L and discharge NT-proBNP cut-off value at 350 ng/L were determined. The cTn I level more than 24 ng/L and NT-proBNP level more than 350 ng/L are associated with increased risk for mortality and readmission (p < 0.01). The mortality and HF-related readmission was significantly increased in patients with high cTn I + high NT-proBNP (p < 0.05), high cTn I + low NT-proBNP (p < 0.05), and low cTn I + high NT-proBNP (p < 0.0%). The increased cTn I or increased NT-proBNP measured in the first 5 consecutive days were significantly associated with 60-day HF-related events (p < 0.05), but the serial measurements did not have a predictive value of 1-year HF outcome. CONCLUSION: This study demonstrates that elevations of discharge cTn I and NT-proBNP are associated with increased 1-year mortality and HF-related readmission. Patients with increasing serial cTnI and NT-proBNP had increased risk for 60-day HF-related events. The two markers can act as independent predicators, and complete each other in prognostic utility of HF patients.

Novel L-lactic acid biosensors based on conducting polypyrrole-block copolymer nanoparticles.

The development of advanced nanomaterials for the highly efficient electrical detection of biological species has attracted great attention. Here, novel polypyrrole-Pluronic F127 nanoparticles (PPy-F127 NPs) with conducting and biocompatibility properties were synthesized and used to construct a L-lactic acid biosensor that could be applied in biochemical assays. The PPy-F127 NPs were characterized by transmission electron microscopy (TEM), elemental analysis and UV-vis spectroscopy. Lactate oxidase (LOx) structure variation on the PPy-F127 NPs was investigated by circular dichroism (CD). The cyclic voltammetric results indicated that LOx immobilized on the PPy-F127 NPs exhibited direct electron transfer reaction with a formal potential value (E(0)') of 0.154 V vs. SCE. Moreover, the biosensor had good electrocatalytic activity toward L-lactic acid with a wide linear range (0.015-37.5 mM) and a low detection limit of 0.0088 mM. The regression equation was I (µA) = 0.02353c (mM) + 1.4135 (R(2) = 0.9939). The L-lactic acid biosensor had a good anti-interference property towards uric acid (UA), ascorbic acid (AA), glucose and cysteine. The idea and method provide a promising platform for the rapid development of biosensors that can be used in the detection of biological species.

Transcriptomic profiling revealed the regulatory mechanism of Arabidopsis seedlings response to oxidative stress from cryopreservation.

KEY MESSAGE: Elevated antioxidant status and positive abiotic stress response in dehydration enhance cell resistance to cryoinjury, and controlling oxidative damage via reactive oxygen species homeostasis maintenance leads to high survival. Cryoprotectants are important for cell survival in cryopreservation, but high concentrations can also cause oxidative stress. Adding vitamin C to the cryoprotectant doubled the survival ratio in Arabidopsis thaliana 60-h seedlings (seedlings after 60-h germination) cryopreservation. In this study, the metabolites and transcriptional profiling of 60-h seedlings were analyzed in both the control cryopreservation procedure (CCP) and an improved cryopreservation procedure (ICP) to reveal the mechanism of plant cell response to oxidative stress from cryopreservation. Reactive oxygen species (ROS) and peroxidation levels reached a peak after rapid cooling-warming in CCP, which were higher than that in ICP. In addition, gene regulation was significantly increased in CCP and decreased in ICP during rapid cooling-warming. Before cryogenic treatment, the number of specifically regulated genes was nearly 10 times higher in ICP dehydration than CCP dehydration. Among these genes, DREBs/CBFs were beneficial to cope with cryoinjury, and calcium-binding protein, OXI1, WRKY and MYB family members as key factors in ROS signal transduction activated the ROS-producing and ROS-scavenging networks including AsA-GSH and GPX cycles involved in scavenging H2O2. Finally, elevated antioxidant status and oxidative stress response in the improved dehydration enhanced seedling resistance to cryogenic treatment, maintained ROS homeostasis and improved cell recovery after cryopreservation.

ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox.

KEY MESSAGE: Oxidative stress and apoptosis-like programmed cell death, induced in part by H 2 O 2 , are two key factors that damage cells during plant cryopreservation. Their inhibition can improve cell viability. We hypothesized that oxidative stress and apoptosis-like event induced by ROS seriously impact plant cell viability during cryopreservation. This study documented changes in cell morphology and ultrastructure, and detected dynamic changes in ROS components (O 2 (·-) , H2O2 and OH·), antioxidant systems, and programmed cell death (PCD) events during embryonic callus cryopreservation of Agapanthus praecox. Plasmolysis, organelle ultrastructure changes, and increases in malondialdehyde (a membrane lipid peroxidation product) suggested that oxidative damage and PCD events occurred at several early cryopreservation steps. PCD events including autophagy, apoptosis-like, and necrosis also occurred at later stages of cryopreservation, and most were apoptosis. H2O2 is the most important ROS molecule mediating oxidative damage and affecting cell viability, and catalase and AsA-GSH cycle are involved in scavenging the intracellular H2O2 and protecting the cells against stress damage in the whole process. Gene expression studies verified changes of antioxidant system and PCD-related genes at the main steps of the cryopreservation process that correlated with improved cell viability. Reducing oxidative stress or inhibition of apoptosis-like event by deactivating proteases improved cryopreserved cell viability from 49.14 to 86.85 % and 89.91 %, respectively. These results verify our model of ROS-induced oxidative stress and apoptosis-like event in plant cryopreservation. This study provided a novel insight into cell stress response mechanisms in cryopreservation.

Cryopreservation affects ROS-induced oxidative stress and antioxidant response in Arabidopsis seedlings.

Plant recovery status after cryopreservation by vitrification had a negative relationship to the oxidative stress induced by reactive oxygen species (ROS). Arabidopsis thaliana seedlings germinated for 48 h or 72 h with different survival tolerances were examined at five steps of cryopreservation, to determine the role of ROS (O2(-), H2O2 and OH) and antioxidant systems (SOD, POD, CAT, AsA and GSH) in cryo-injury. In addition, the effects of the steps on membrane lipid peroxidation were studied using malondialdehyde (MDA) as an indicator. The results indicated that H2O2-induced oxidative stress at the steps of dehydration and rapid warming was the main cause of cryo-injury of 48-h seedlings (high survival rate) and 72-h seedlings (no survival). The H2O2 was mainly generated in cotyledons, shoot tips and roots of seedlings as indicated by Amplex Red staining. Low survival of 72-h seedlings was associated with severe membrane lipid peroxidation, which was caused by increased OH generation activity and decreased SOD activity. The antioxidant-related gene expression by qRT-PCR and physiological assays suggested that the antioxidant system of 48-h seedlings were activated by ROS, and they mounted a defense against oxidative stress. A high level of ROS led to the weakening of the antioxidant system of 72-h seedlings. Correlation analysis indicated that enhanced antioxidant enzymes activities contributed to the high survival rate of 48-h seedlings, which could reflect by cryopreservation of antioxidant mutant seedlings. This model system indicated that elevated CAT activity and AsA content were determinants of cryogenic stress tolerance, whose manipulation could improve the recovery of seedlings after cryopreservation.