Evaluation of Thellungiella halophila ST7 for improving salt tolerance in cotton

Background Gossypium hirsutum (upland cotton) is one of the principal fiber crops in the world. Cotton yield is highly affected by abiotic stresses, among which salt stress is considered as a major problem around the globe. Transgenic approach is efficient to improve cotton salt tolerance but depending on the availability of salt tolerance genes. Results In this study we evaluated salt tolerance candidate gene ST7 from Thellungiella halophila, encoding a homolog of Arabidopsis aluminum-induced protein, in cotton. Our results showed that ThST7 overexpression in cotton improved germination under NaCl stress as well as seedling growth. Our field trials also showed that ThST7 transgenic cotton lines produced higher yield under salt stress conditions. The improved salt tolerance of the transgenic cotton lines was partially contributed by enhanced antioxidation as shown by diaminobenzidine (DAB) and nitrotetrazolium blue chloride (NBT) staining. Moreover, transcriptomic analysis of ThST7 overexpression lines showed a significant upregulation of the genes involved in ion homeostasis and antioxidation, consistent with the salt tolerance phenotype of the transgenic cotton. Conclusions Our results demonstrate that ThST7 has the ability to improve salt tolerance in cotton. The ThST7 transgenic cotton may be used in cotton breeding for salt tolerance cultivars.

Fourier transform infrared spectroscopy coupled with machine learning classification for identification of oxidative damage in freeze-dried heart valves

Freeze-drying can be used to ensure off-the-shelf availability of decellularized heart valves for cardiovascular surgery. In this study, decellularized porcine aortic heart valves were analyzed by nitroblue tetrazolium (NBT) staining and Fourier transform infrared spectroscopy (FTIR) to identify oxidative damage during freeze-drying and subsequent storage as well as after treatment with H2O2 and FeCl3. NBT staining revealed that sucrose at a concentration of at least 40% (w/v) is needed to prevent oxidative damage during freeze-drying. Dried specimens that were stored at 4 °C depict little to no oxidative damage during storage for up to 2 months. FTIR analysis shows that fresh control, freeze-dried and stored heart valve specimens cannot be distinguished from one another, whereas H2O2- and FeCl3-treated samples could be distinguished in some tissue section. A feed forward artificial neural network model could accurately classify H2O2 and FeCl3 treated samples. However, fresh control, freeze-dried and stored samples could not be distinguished from one another, which implies that these groups are very similar in terms of their biomolecular fingerprints. Taken together, we conclude that sucrose can minimize oxidative damage caused by freeze-drying, and that subsequent dried storage has little effects on the overall biochemical composition of heart valve scaffolds.

The Rice LRR-Like1 Protein YELLOW AND PREMATURE DWARF 1 is Involved in High Light-Induced Leaf Senescence

Senescence is induced by endogenous physiological changes or exogenous stresses in plants. Here, we isolated two alleles of a novel rice (Oryza sativa L.) mutant, yellow and premature dwarf 1 (ypd1). The ypd1 mutants exhibited a yellow and dwarf phenotype from germination, and premature senescence starting at tillering. Moreover, the ypd1 mutants were sensitive to high light, which accelerated cell death and senescence. Consistent with their yellow phenotype, the ypd1 mutants had abnormal chloroplasts and lower levels of photosynthetic pigments. Trypan blue staining, TUNEL experiments, NBT staining, and DAB staining demonstrated that the ypd1 mutants showed cell death and accumulated reactive oxygen species. Moreover, the ypd1 mutants showed increased expression of senescence-associated genes. Map-based cloning revealed a substitution (G→A) in exon 6 (ypd1-1) and 13 (ypd1-2) of LOC_Os06g13050 that affected splicing and caused premature termination of the encoded protein. YPD1 was preferentially expressed in the leaf and encodes an LRR-Like1 (LRRL1) protein. Complementation, overexpression, and targeted deletion experiments confirmed that the mutations in YPD1 cause the ypd1 phenotype. YPD1 localized on the chloroplast membrane. These findings revealed that the novel rice LRRL1 protein YPD1 affects rice chloroplast development and leaf senescence.

Advantages and Limitations of Salmon-Gal/Tetrazolium Salt Histochemistry for the Detection of LacZ Reporter Gene Activity in Murine Epithelial Tissue

The Escherichia coli LacZ gene is a widely used reporter for gene regulation studies in transgenic mice. It encodes bacterial β-galactosidase (Bact β-Gal), which causes insoluble precipitates when exposed to chromogenic homologues of galactose. We and others have recently reported that Bact β-Gal detection with Salmon-Gal (S-Gal) in combination with nitro blue tetrazolium chloride (NBT) is very sensitive and not prone to interference by acidic endogenous β-galactosidases. Unfortunately, as we show here, the method appears to be inadequate for evaluation of Bact β-Gal expression in keratinized epithelial appendages but not in other keratinized epithelia. NBT in the reaction mixture, just as other tetrazolium salts, inevitably causes unwanted staining artifacts in lingual filiform papillae, penile spines, and hair fibers by interacting with keratin sulfhydryl-rich regions. The methodological limitation can be overcome in part by pretreating the tissues before the S-Gal/NBT staining with an iodine–potassium iodide solution. Alternatively, the use of iodonitrotetrazolium chloride instead of NBT in the S-Gal reaction mixture provides enough color resolution to distinguish the specific Bact β-Gal staining in orange from the artifact staining in dark red. In summary, we provide evidence that S-Gal/NBT histochemistry has limitations, when staining keratinized epithelial appendages.

Antileukemic Potential ofMomordica charantiaSeed Extracts on Human Myeloid Leukemic HL60 Cells

Momordica charantia(bitter gourd) has been used in the traditional system of medicine for the treatment of various diseases. Anticancer activity ofM. charantiaextracts has been demonstrated by numerousin vitroandin vivostudies. In the present study, we investigated the differentiation inducing potential of fractionatedM. charantiaseed extracts in human myeloid HL60 cells. We found that the HL60 cells treated with the fractionated seed extracts differentiated into granulocytic lineage as characterized by NBT staining, CD11b expression, and specific esterase activity. The differentiation inducing principle was found to be heat-stable, and organic in nature. The differentiation was accompanied by a downregulation ofc-myctranscript, indicating the involvement ofc-mycpathway, at least in part, in differentiation. Taken together these results indicate that fractionated extracts ofM. charantiaseeds possess differentiation inducing activity and therefore can be evaluated for their potential use in differentiation therapy for leukemia in combination with other inducers of differentiation.

Heterogeneous distribution of a fatty acid analogue uptake in the myocardium of aged rats

The aim of this study was to determine the extent and location of damaged myocardial areas in senescent rats. The viability of myocardial cells was evaluated in virgin young (4 months old) and aged (29 months old) female Wistar rats by analysing the uptake of a slowly metabolisable radiolabelled fatty acid analogue, 15-p-iodophenyl-β-methylpentadecanoic acid (IMPPA). The biodistribution of IMPPA was measured in various organs, and regional myocardial uptake was specifically assessed using quantitative autoradiography. Myocardial enzymatic activity and DNA content were also evaluated with nitro blue tetrazolium (NBT) and propidium iodide (PÏ) staining, respectively. In senescent rats, cardiac and renal IMPPA uptake showed a significant (50%) reduction compared with young adult rats and the uptake was not significantly changed in the liver, spleen, lungs, and skeletal muscle. Total ventricular NBT staining and IMPPA uptake were almost homogeneous in young adult rats, whereas they were very heterogeneous in aged rats. In the latter, approximately 11% of the total ventricular volume showed a significantly decreased (by 60% or more) IMPPA uptake compared with normal values, and this reduction was greater in ventricle base than in apex. The myocardial areas unlabelled or poorly labelled by IMPPA represented 4, 5, 6, and 21% of the right ventricular, left ventricular epicardial, septal, and left ventricular endocardial volume, respectively, and were poorly stained with NBT. In some of these areas, PI staining indicated the presence of living cells unable to pick up NBT staining. In conclusion, in young adult rats, no myocardial lesions were observed using three different labelling techniques. However, important and significant myocardial lesioned areas were detected in senescent rats and were located preferentially in the left ventricular endocardium, as shown by a decrease in NBT staining and IMPPA uptake. These likely corresponded to a reduced number of cardiomyocytes and (or) a reduced aerobic substrate utilization, along with the development of fibrosis.Key words: senescent female rats, fatty acid analogue uptake, myocyte loss, lesioned and borderline areas.