Direct investigations of the effects of nicardipine on calcium channels of astrocytes by Atomic Force Microscopy.

Calcium channel blockers (CCB) of astrocytes can blockade the calcium ions entry through the voltage gated calcium channels (VGCC), and is widely used in the diseases related with VGCC of astrocytes. But many aspects of the interaction mechanisms between the CCB and VGCC of astrocytes still remain unclear due to the limited resolution of the approaches. Herein the effects of the nicardipine (a type of CCB) on VGCC of astrocytes were investigated at very high spatial, force and electrical resolution by multiple modes of Atomic Force Microscopy (AFM) directly. The results reveal that after the addition of nicardipine, the recognition signals of VGCC disappeared; the specific unbinding forces vanished; the conductivity of the astrocytes decreased (the current decreased about 2.9 pA and the capacitance was doubled); the surface potential of the astrocytes reduced about 14.2 mV. The results of electrical properties investigations are consistent with the simulation experiments. The relations between these biophysical and biochemical properties of VGCC have been discussed. All these demonstrate that the interactions between nicardipine and VGCC have been studied at nanometer spatial resolution, at picoNewton force resolution and very high electrical signal resolution (pA in current, pF in capacitance and 0.1 mV in surface potential) level. The approaches are considered to be high resolution and high sensitivity, and will be helpful and useful in the further investigations of the effects of other types of CCB on ion channels, and will also be helpful in the investigations of mechanisms and therapy of ion channelopathies.

Atomic force microscopy correlates mechanical and electrical properties of HepG2 cells with curcumin concentration.

Hepatocellular carcinoma (HCC) is a highly prevalent cancer with a significant impact on human health. Curcumin, a natural compound, induces cytoskeletal changes in liver cancer cells and modifies the distribution of lipids, proteins, and polysaccharides on plasma membranes, affecting their mechanical and electrical properties. In this study, we used nanomechanical indentation techniques and Kelvin probe force microscopy (KPFM) based on atomic force microscopy (AFM) to investigate the changes in surface nanomechanical and electrical properties of nuclear and cytoplasmic regions of HepG2 cells in response to increasing curcumin concentrations. CCK-8 assays and flow cytometry results demonstrated time- and concentration-dependent inhibition of HepG2 cell proliferation by curcumin. Increasing curcumin concentration led to an initial increase and then decrease in the mechanical properties of nuclear and cytoplasmic regions of HepG2 cells, represented by the Young's modulus (E), as observed through nanoindentation. KPFM measurements indicated decreasing trends in both cell surface potential and height. Fluorescence microscopy results indicated a positive correlation between curcumin concentration and phosphatidylserine translocation from the inner to the outer membrane, which influenced the electrical properties of HepG2 cells. This study provides valuable insights into curcumin's mechanisms against cancer cells and aids nanoscale evaluation of therapeutic efficacy and drug screening.

Single molecule localizations of voltage-gated sodium channel NaV1.5 on the surfaces of normal and cancer breast cells.

Voltage-gated sodium channels (VGSCs) are widely expressed in various types of tumor and cancer cells, and NaV1.5 is overexpressed in highly metastatic breast cancer cells. There may be positive relations between the expression levels of NaV1.5 and breast cancer recurrence and metastasis. Herein, NaV1.5 was detected and localized on the surfaces of normal and cancer breast cells by the single molecule recognition imaging (SMRI) mode of atomic force microscopy (AFM). The results reveal that NaV1.5 was irregularly distributed on the surfaces of normal and cancer breast cells. The NaV1.5 has an area percentage of 0.6% and 7.2% on normal and cancer breast cells, respectively, which indicates that there is more NaV1.5 on cancer cells than on normal cells. The specific interaction forces and binding kinetics in the NaV1.5-antibody complex system were investigated with the single molecule force spectroscopy (SMFS) mode of AFM, indicating that the stability of the NaV1.5-antibody on normal breast cells is higher than that on cancer breast cells. All these results will be useful to study the interactions of other ion channel-antibody systems, and will also be useful to understand the role of sodium channels in tumor metastasis and invasion.

Injectable adipose-derived stem cells-embedded alginate-gelatin microspheres prepared by electrospray for cartilage tissue regeneration.

Objective: To prepare adipose-derived stem cells (ADSCs)-embedded alginate-gelatinemicrospheres (Alg-Gel-ADSCs MSs) by electrospray and evaluate their feasibility for cartilage tissue engineering. To observe the efficacy of Alg-Gel-ADSCs MSs in repairing articular cartilage defects in SD rats. Methods: ADSCs were isolated and characterized by performing induced differentiation and flow cytometry assays. Alginate-gelatine microspheres with different gelatine concentrations were manufactured by electrospraying, and the appropriate alginate-gelatine concentration and ratio were determined by evaluating microsphere formation. Alg-Gel-ADSCs MSs were compared with Alg-ADSCs MSs through the induction of chondrogenic differentiation and culture. Their feasibility for cartilage tissue engineering was analysed by performing Live/Dead staining, cell proliferation analysis, toluidine blue staining and a glycosaminoglycan (GAG) content analysis. Alg-Gel-ADSCs MSs were implanted in the cartilage defects of SD rats, and the cartilage repair effect was evaluated at different time points. The evaluation included gross observations and histological evaluations, fluorescence imaging tracking, immunohistochemical staining, microcomputed tomography (micro-CT) and a CatWalk evaluation. Results: The isolated ADSCs showed multidirectional differentiation and were used for cartilage tissue engineering. Using 1.5 w:v% alginate and 0.5 w:v% gelatine (Type B), we successfully prepared nearly spherical microspheres. Compared with alginate microspheres, alginate gel increased the viability of ADSCs and promoted the proliferation and chondrogenesis of ADSCs. In our experiments on knee cartilage defects in SD rats in vivo, the Alg-Gel-ADSCs MSs showed superior cartilage repair in cell resides, histology evaluation, micro-CT imaging and gait analysis. Conclusions: Microspheres composed of 1.5 w:v% alginate-0.5 w:v% gelatine increase the viability of ADSCs and supported their proliferation and deposition of cartilage matrix components. ADSCs embedded in 1.5 w:v% alginate-0.5 w:v% gelatine microspheres show superior repair efficacy and prospective applications in cartilage tissue repair. The translational potential of this article: In this study, injectable adipose-derived stem cells-embedded alginate-gelatin microspheres (Alg-Gel-ADSCs MSs) were prepared by the electrospray . Compared with the traditional alginate microspheres, its support ability for ADSCs is better and shows a better repair effect. This study provides a promising strategy for cartilage tissue regeneration.

Nitrate removal performances of a new aerobic denitrifier, Acinetobacter haemolyticus ZYL, isolated from domestic wastewater.

A new aerobic denitrifying bacterium ZYL was isolated from domestic wastewater sludge and identified as Acinetobacter haemolyticus (similarity 99%) by the 16S rDNA sequencing analysis. The strain could use nitrate, nitrite and ammonium as the sole N-source for growth with a final product of N2, demonstrating its good abilities for aerobic denitrification and heterotrophic nitrification. Single-factor experiment results showed that the effective removal of nitrate by strain ZYL occurred with carbon source sodium succinate, C/N 16-24, pH 5-9, temperature 20-40 °C, DO ≥ 4.84 mg/L. Ammonium was preferentially used by strain ZYL with nitrate and ammonium as the mixed nitrogen sources. According to nitrogen utilization, nitrogen balance analysis, enzyme assay and denitrifying gene amplification, nitrate was assimilated directly by the isolate for cell synthesis and also converted into N2 through aerobic denitrification. All these make strain ZYL an ideal choice for treating nitrogen-containing wastewater.

Cloning and functional identification of a Chilo suppressalis-inducible promoter of rice gene, OsHPL2.

BACKGROUND: Promoters play a key role in driving insect-resistant genes during breeding of transgenic plants. In current transgenic procedures for breeding rice resistance to striped stem borer (Chilo suppressalis Walker, SSB), the constitutive promoter is used to drive the insect-resistant gene. To reduce the burden of constitutive promoters on plant growth, isolation and identification of insect-inducible promoters are particularly important. However, few promoters are induced specifically by insect feeding. RESULTS: We found rice hydroperoxide lyase gene (OsHPL2) (LOC_Os02g12680) was upregulated after feeding by SSB. We subsequently cloned the promoter of OsHPL2 and analysed its expression pattern using the ß-glucuronidase (GUS) reporter gene. Histochemical assays and quantitative analyses of GUS activity confirmed that P HPL2 :GUS was activated by SSB, but did not respond to brown planthopper (Nilaparvata lugens Stål, BPH) infestation, mechanical wounding or phytohormone treatments. A series of 5' truncated assays were conducted and three positive regulatory regions (-1452 to -1213, -903 to -624, and -376 to -176) induced by SSB infestation were identified. P2R123-min 35S and P2TR2-min 35S promoters linked with cry1C of transgenic plants showed the highest levels of Cry1C protein expression and SSB larval mortality. CONCLUSION: We identified an SSB-inducible promoter and three positive internal regions. Transgenic rice plants with the OsHPL2 promoter and its positive regions driving cry1C exhibited the expected larvicidal effect on SSB. Our study is the first report of an SSB-inducible promoter that could be used as a potential resource for breeding insect-resistant transgenic crops. © 2020 Society of Chemical Industry.

Assessing the effects of Cry2Aa protein on Habrobracon hebetor (Hymenoptera: Braconidae), a parasitoid of Indian meal moth, Plodia interpunctella (lepidoptera: Pyralidae).

Transgenic crops express Cry proteins exhibit high resistant to target insect pests. When we evaluate the effects of Cry proteins on the parasitoid of target insect pest via tritrophic experiments (transgenic plant-target insect pest-parasitoid) host quality of parasitoids might decrease because of insecticidal protein ingestion, this would cause host-quality mediated effects and influence the accuracy of biosafety assessment. In the current study, high dose of Cry2Aa protein was injected into the hemolymph of Plodia interpunctella by microinjection, and the hemolymph was used as the carrier to deliver Cry protein to Habrobracon hebetor, which has been previously reported as an ectoparasitoid of P. interpunctella larval, in order to avoid the "host-quality mediated effects". Results showed that injected Cry2Aa remained at high concentration and bioactive in the hemolymph of P. interpunctella parasitized by H. hebetor, the hemolymph of P. interpunctella could be used as carriers of Cry protein to H. hebetor, and high dose of Cry2Aa have no negative impacts on the development time, weight of pupa, sex ratio, adults weight (male and female), adult longevity and fecundity, and the activity of stress-related enzymes of H. hebetor. However, the hemolymph of P. interpunctella injected into Galanthus nivalis agglutinin (the positive control) showed significant negative impact on these parameters measured in the present study of H. hebetor. This indicated that Cry2Aa protein had no detrimental effects on the biological parameters of H. hebetor measured in the current study. Meanwhile, this study provides a new method for the safety evaluation of the ectoparasitoids of target pest and might be expanded to the other species of ectoparasitoids of target insects of Cry proteins in biosafety risk assessment.

Identification, characterization, and expression analysis of auxin response factor (ARF) gene family in Brachypodium distachyon.

Auxin response factors (ARFs) are one type of essential family of transcription factors that bind with auxin response elements (AuxRE), and play vital roles in variety of plant development and physiological processes. Brachypodium distachyon, related to the major cereal grain species, were recently developed to be a good model organism for functional genomics research. So far, genome-wide overview of the ARF gene family in B. distachyon was not available. Here, a systemic analysis of ARF gene family members in B. distachyon was performed. A comprehensive overview of the characterization of the BdARFs was obtained by multiple bioinformatics analyses, including the gene and protein structure, chromosome locations, conserved motifs of proteins, phylogenetic analysis, and cis-elements in promoters of BdARF. Results showed that all BdARFs contained conserved DBD, MR, and CTD could be divided into four classes, Ia, IIa, IIb, and III. Expression profiles of BdARF genes indicated that they were expressed across various tissues and organs, which could be clustered into three main expression groups, and most of BdARF genes were involved in phytohormone signal transduction pathways and regulated physiological process in responding to multiple environmental stresses. And predicted regulatory network between B. distachyon ARFs and IAAs was also discussed. Our genomics analysis of BdARFs could yield new insights into the complexity of the control of BdARF genes and lead to potential applications in the investigation of the accurate regulatory mechanisms of ARFs in herbaceous plants.

Knockdown of TOR causing ovarian diapause in a genetically stable brachypterous strain of Nilaparvata lugens.

Brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is one of the most damaging pests of rice crops. BPH is a migratory insect with a delayed ovarian development in migrants classified as reproductive diapause. The molecular mechanism of reproductive diapause remains unclear, although we suspect it might be regulated by one or more nutrient signaling pathways. The target of rapamycin (TOR) pathway regulates cell growth in response to nutritional information, which raised a hypothesis that TOR mediates BPH reproductive diapause. We used a pure brachypterous strain (BS) and a predominantly macropterous strain (MS) to investigate the roles of NlTOR in BPH reproductive diapause. We found that NlTOR is expressed from the nymphal to adult stages, with a higher expression level of NlTOR in BS adults at 1, 2, and 4 days posteclosion than in MS at the same time points. Injection of dsNlTOR into BS nymphs resulted in the termination of BPH female ovary development and the retardation of nymph development. We infer that TOR signaling functions in BPH reproductive diapause by regulating the expression of NlFoxA and NlVitellogenin.

The tiered-evaluation of the effects of transgenic cry1c rice on Cyrtorhinus lividipennis, a main predator of Nilaparvata lugens.

T1C-19, a newly developed transgenic cry1C rice line, expresses cry1C under the control of the maize ubiquitin promoter, and is highly resistant to lepidopteran pests of rice. Cyrtorhinus lividipennis is the major predator of the eggs and young nymphs of Nilaparvata lugens, which is the main non-target sap-sucking insect pest of Bt rice. C. lividipennis may be exposed to Cry1C protein, thus biosafety evaluations of transgenic cry1C rice on C. lividipennis should be conducted before the commercialization of T1C-19. In the current study, we tested the direct toxicity of elevated doses of Cry1C to C. lividipennis, effects of T1C-19 on the life-table parameters of C. lividipennis via preying planthoppers, and effects of T1C-19 on the population density and dynamics in rice fields. No detrimental effects on development, survival, female ratio and body weight of C. lividipennis were caused by direct exposure to elevated doses of the Cry1C protein or prey-mediated exposure to realistic doses of the protein. The population density and dynamics did not significantly differ between C. lividipennis in T1C-19 and non-transgenic rice fields. Thus, transgenic cry1C rice had no negative effects on C. lividipennis. This is the first report of the effects of transgenic cry1C rice on C. lividipennis.

Apterous A modulates wing size, bristle formation and patterning in Nilaparvata lugens.

Apterous A (apA), a member of the LIM-homeobox gene family, plays a critical role in the development of wing. The achaete-scute Complex (AS-C) encodes basic helix-loop-helix (bHLH) transcription factors and functions in bristle development. In the present study, we cloned apA (NlapA) and an achaete-scute homologue (NlASH) from N. lugens. Levels of NlapA and NlASH were higher in nymphs than adults, with particularly high expression in the thorax of nymphs. NlapA expressed more highly in nymphs of the macropterous strain (MS) than those of the brachypterous strain (BS) at 2(nd) and 4(th) instar. Knockdown of NlapA and NlASH in vivo generated similar phenotypic defects in the wing (loss-of-bristles, twisted or erect wing). Silencing of NlapA in nymphs of MS led to decreased wing size in adults. Moreover, depletion of NlapA suppressed expression of NlDl, Nlsal, Nlser, Nlvg and Nlwg, both in MS and BS, but induced differential responses of Nlubx and Nlnotch expression between MS and BS. Notably, expression of NlASH was regulated by NlapA. These results collectively indicate that NlapA is an upstream modulator of wing size, bristle formation and patterning. Further studies on DNA-protein and protein-protein interactions are required to elucidate NlapA-mediated regulation of wing development.

Transgenic Amorphophallus konjac expressing synthesized acyl-homoserine lactonase (aiiA) gene exhibit enhanced resistance to soft rot disease.

Amorphophallus konjac is an important economic crop widely used in health products and biomaterials. However, this monocotyledonous plant's production is seriously restricted by soft rot disease. Some Bacillus thuringiensis strains generate an endocellular acyl homoserine lactonase (AiiA), which has inhibitory effect on soft rot pathogen through disrupting the signal molecules (N-acylhomoserine lactones, AHL) of their Quorum Sensing system. The aim of our study is to obtain transgenic A. konjac expressing AiiA protein and exhibiting resistance to soft rot. But till now, there is not any report about exogenous gene transformation in A. konjac. In this research, an Agrobacterium-mediated genetic transformation system was constructed. An aiiA gene was synthesized according to the codon usage in A. konjac. Embryogenic callus was infected with the A. tumefaciens strain EHA105 harboring the plant transformation plasmid pU1301 plus synthesized aiiA gene. After antibiotics screening, 34 plants were obtained. PCR analysis showed that positive amplified fragments were present in 21 out of these 34 lines. Southern blot analysis indicated that aiiA gene had integrated into the genome of A. konjac. Western blotting demonstrated that the target protein of interest was reactive with the antibody against AiiA. Further disease resistance detection revealed that all of the tested transgenic A. konjac lines exhibited high resistance to soft rot bacteria Erwinia carotovora subsp. Carotovora (Ecc) SCG1. The protocol is useful for the quality improvement of A. konjac through genetic transformation.