Development of a protoplast isolation system for functional gene expression and characterization using petals of Camellia Oleifera.

Protoplasts preparation and purification have been frequently used in plant genetics and breeding studies, whereas application of protoplasts in woody plants is still in its infancy. Although transient gene expression using purified protoplasts is well-documented and widely used in model plants and agriculture crops, no instance of either stable transformation or transient gene expression in the woody plant Camellia Oleifera has as of yet been reported. Here, we developed a protoplast preparation and purification method using C. oleifera petals by optimizing osmotic condition with D-mannitol and polysaccharide-degrading enzyme concentrations for petal cell wall digestion, to reach a high efficiency of protoplast productivity and viability. The achieved protoplasts yield was approximately 1.42 × 107 cells per gram of petal material and the viability of protoplasts was up to 89%. In addition, we explored influencing factors of protoplast transformation, including concentrations of PEG4000 and plasmid DNA. The transformation efficiency of 81% could be reached under the optimized condition. This protoplast isolation and transient expression system were deployed to further identify the functional regulation of C. oleifera related genes and the subcellular distribution of their encoded products. In summary, the protoplast isolation and transient expression system we established using oil-tea tree petals is an efficient, versatile and time-saving system, being suitable for gene function characterization and molecular mechanism analysis.

Changes of Electrocardiogram and Myocardial Enzymes in Patients with Intracerebral Hemorrhage.

Purpose: Cardiac complications are common in patients with spontaneous intracerebral hemorrhage (ICH). The present study is aimed at observing the incidence of cardiac complications after ICH, so as at improving the understanding of the relationship between cardiac complications and ICH. Methods: This is a retrospective study on analyzing electrocardiogram (ECG) and serum myocardial enzyme of 208 patients with ICH admitted to a tertiary hospital from 2018 to 2019. For each patient, demographics, medical history, clinical presentation, ECG, serum myocardial enzyme, and head CT on admission were reviewed. Mortality was noted. Results: Among the 208 patients, 145 (69.71%) had one or more ECG abnormalities. The top three abnormalities were corrected QT interval (QTc) prolongation 52 (25%), ST depression 48 (23.08%), and T wave inversion 38 (18.27%). One hundred and thirty-nine patients (66.83%) had increased serum levels of at least one kind of myocardial enzyme, which were high-sensitive cardiac troponin T (hs-cTnT) 79 (37.98%), lactic dehydrogenase (LDH) 80 (38.46%), creatine kinase (CK) 57 (27.40%), and creatine kinase-myocardial subfraction (CKMB) 57 (27.40%). The logistic regression analysis showed the following: secondary intraventricular hemorrhage (SIVH) (odds ratio (OR) 5.32; 95% confidence interval (CI) 2.55-11.08; p < 0.001) and hematoma volume > 30 ml (OR 3.81; 95% CI 1.86-7.81; p < 0.001) were independent predictive factors of QTc prolongation; thalamus location (OR 5.79; 95% CI 1.94-17.28; p < 0.05), hematoma volume > 30 ml (OR 24.187; 95% CI 3.14-186.33; p < 0.05), insular involvement (OR 19.08; 95% CI 5.77-63.07; p < 0.001), and SIVH (OR 2.62; 95% CI 1.69-5.86; p < 0.05) were independent predictive factors of ST depression; insular involvement (OR 2.90; 95% CI 1.12-7.50; p < 0.05) and hematoma volume > 30 ml (OR 1.98; 95% CI 1.06-3.70; p < 0.05) were independent predictive factors of increase of CK; Glasgow Coma Scale (GCS) (OR 0.86; 95% CI 0.78-0.98; p < 0.05) and insular involvement (OR 5.56; 95% CI 1.98-15.62; p < 0.05) were independent predictive factors of increase of CKMB; SIVH (OR 2.05; 95% CI 1.07-3.92; p < 0.05) was independent predictive factor of increase of LDH; age (OR 1.03; 95% CI 1.01-1.06; p < 0.05), blood glucose on admission (OR 1.10; 95% CI 1.01-1.20; p < 0.05), and history of antiplatelet drug use (OR 3.50; 95% CI 1.01-12.12; p < 0.05) were independent predictive factors of hs-cTnT. All the injury indexes were not related to in-hospital mortality. Conclusion: The study suggests that insular involvement, hematoma volume > 30 ml, and SIVH are the strongest risk factors for ECG abnormalities and elevated myocardial enzymes after ICH followed which are the risk factors such as GCS, age, admission blood glucose, and ICH location in the thalamus.

An Efficient Modular Gateway Recombinase-Based Gene Stacking System for Generating Multi-Trait Transgenic Plants.

Transgenic technology can transfer favorable traits regardless of reproductive isolation and is an important method in plant synthetic biology and genetic improvement. Complex metabolic pathway modification and pyramiding breeding strategies often require the introduction of multiple genes at once, but the current vector assembly systems for constructing multigene expression cassettes are not completely satisfactory. In this study, a new in vitro gene stacking system, GuanNan Stacking (GNS), was developed. Through the introduction of Type IIS restriction enzyme-mediated Golden Gate cloning, GNS allows the modular, standardized assembly of target gene expression cassettes. Because of the introduction of Gateway recombination, GNS facilitates the cloning of superlarge transgene expression cassettes, allows multiple expression cassettes to be efficiently assembled in a binary vector simultaneously, and is compatible with the Cre enzyme-mediated marker deletion mechanism. The linked dual positive-negative marker selection strategy ensures the efficient acquisition of target recombinant plasmids without prokaryotic selection markers in the T-DNA region. The host-independent negative selection marker combined with the TAC backbone ensures the cloning and transfer of large T-DNAs (>100 kb). Using the GNS system, we constructed a binary vector containing five foreign gene expression cassettes and obtained transgenic rice carrying the target traits, proving that the method developed in this research is a powerful tool for plant metabolic engineering and compound trait transgenic breeding.

Targeted Therapy of Tuberculous Meningitis in Rats with Methylprednisolone Composite Nanoparticles.

It was to investigate the targeted therapeutic effect of methylprednisolone (MPS) composite nanoparticles (NPs) on tuberculous meningitis (TBM) in rats. A total of 180 special pathogen-free (SPF) Sprague Dawley (SD) rats (male) were randomly and equally assigned to the normal control group, TBM infection group, and TBM treatment group. Those in the TBM infection group and the TBM treatment group were injected with Mycobacterium tuberculosis suspension via the tail vein. After the TBM model was established, rats in the TBM treatment group were injected intraperitoneally with methylprednisolone-nano sterically stabilized liposomes (MPS-NSSLs), and those in the normal control group were injected with an equal amount of normal saline. MPS-NSSLs were prepared, and their quality evaluation, encapsulation rate, drug-lipid ratio, and stability were detected. The particle size distribution of MPS-NSSLs was 95.4 ± 0.7 nm, showing a complete spherical structure, and the encapsulation rate was 91.24 ± 0.27 %, and the drug-lipid ratio was about 0.4. After 7 days of treatment, the water content of brain tissue in the TBM infection group was drastically superior to that in the control group (P<0.05); Evans blue (EB) content in the TBM infection group was dramatically superior to that in the control group (P<0.05). The TBM rat model was successfully established, and this model verified that MPS-NSSLs had the characteristics of high efficiency and low toxicity in the treatment of TBM rats.

Study of Intracranial Hematoma Removal and High Intracranial Pressure Reduction Using a Novel Three-Needle Brain Puncture Technique.

Objective: The present study aimed to evaluate the clinical value of minimally invasive surgery for intracranial hematoma removal and high intracranial pressure (ICP) reduction using a novel three-needle brain puncture technique. Methods: A total of 202 cases with supratentorial hematoma were analyzed, 54 of whom received three-needle brain puncture (study group), and the remaining cases received single-needle (control groups 1 and 2) and two-needle brain puncture (control group 3). The amount of intracranial hematoma removed, changes in ICP, retention time of puncture needle, volume of residual blood, the National Institute of Health Stroke Scale (NIHSS) score, and postoperative survival rate were used as indexes to evaluate patient outcomes. Results: We found that three-needle brain puncture (study group) can remove more intracranial hematoma (P < 0.05) and achieve lower ICP (P < 0.05) than single- and two-needle brain puncture (control group). The needle retention time and volume of residual blood significantly decreased in the study group. Additionally, a statistically significant difference was observed in the NIHSS scores and survival rates between the study and control groups (P < 0.05). Conclusion: These data suggest that three-needle minimally invasive stereotactic puncture can effectively remove hematoma, reduce ICP, decrease the degree of brain damage, and improve prognosis.

Effect of Glucocorticoid Nanoliposomes on Vascular Endothelial Growth Factor Expression in Brain Tissue of Rats with Tuberculous Meningitis.

To investigate the effect of human brain-targeted nanoliposomes encapsulating methylprednisolone sodium succinate on the level of vascular endothelial growth factor (VEGF) in brain tissue of rats with tuberculous meningitis (TBM), the nanoliposome DSPE-125I-AIBZM-MPS was prepared. 180 rats were divided into normal control, TBM infection, and TBM treatment groups. The brain water content, Evans blue (EB) content, VEGF, and the gene and protein expression of receptors (Flt-1, Flk-1) of rats after modeling were measured. The brain water content and EB content in the TBM treatment group were significantly lower than those in the TBM infection group at 4 and 7 days after modeling (P < 0.05). The expression of VEGF and its receptor Flt-1 mRNA in the brain tissue of rats in the TBM infection group was significantly higher than that in the normal control group at 1, 4, and 7 days after modeling (P < 0.05). The expression of VEGF and its receptor Flt-1 mRNA in the brain tissue of rats in the TBM treatment group was significantly higher than that in the TBM infection group at 1, 4, and 7 days after modeling (P < 0.05). In summary, the prepared DSPE-125I-AIBZM-MPS nanoliposomes can effectively reduce brain water content and EB content and reduce the release of inflammatory factors of brain tissue in rats, playing a role in the treatment of TBM in rats by regulating the expression of VEGF and its receptor Flt-1 mRNA.

Construction of introgression lines of Oryza rufipogon and evaluation of important agronomic traits.

Cultivated rice (Oryza sativa) originated from common wild rice (Oryza rufipogon) and inherited its advantages. However, during the rice domestication process, some valuable features of wild rice, such as tolerance to biotic and abiotic stress, were lost. To fully utilize wild rice germplasm resources, we constructed a set of introgression lines (ILs) using a common wild rice material from Lingshui, China. A set of high-resolution InDel molecular markers with an average interval of 2.39 Mb were designed to carry out marker-assisted selection and identification of segment characteristics. The ILs contained 77 lines including 1.286 introgressed fragments with an average length of 6.511 Mb, covering 93.59% of the donor parent's chromosomes. The agricultural traits of 77 lines were investigated. Many old quantitative trait loci (QTLs) involved in plant height, awn length, seed traits and other characteristics reappeared in our ILs, proving that our system was reliable. Further, many new QTLs were identified. A QTL related to drought tolerance located on chromosome 4 was thoroughly elaborated. This set of ILs provides a new resource for utilizing the excellent features of wild rice.

Minimally invasive procedures reduced the damages to motor function in patients with thalamic hematoma: observed by motor evoked potential and diffusion tensor imaging.

BACKGROUND: The purpose of this study was to observe changes in motor function using diffusion tensor imaging (DTI) and motor-evoked potential (MEP) in patients with thalamic hematoma treated by minimally invasive procedures. METHODS: Forty-three patients with thalamic hematoma were randomized to either a minimally invasive group (MI group) or a medical treatment group (MT group). The patients in the MI group underwent whole-brain DTI and MEP measurements both before and 2 weeks after the thalamic hematoma was evacuated by minimally invasive procedures. The fractional anisotropy (FA) values of the corticospinal tract (CST) in the internal capsule and MEP ipsilateral to the hematoma side and the contralateral side were determined and then compared with the MT group. RESULTS: DTI showed that fibers in the internal capsule ipsilateral to the hematoma decreased either in number or were interrupted because of hematoma-induced damages, and in both groups, the CST FA values on admission were significantly lower (0.428 ± 0.032 and 0.415 ± 0.048 for the MI and MT groups, respectively) than the control values. Two weeks after the hematoma was evacuated, the number of fibers and the FA values of the CST in the internal capsule had both increased significantly relative to the values on admission. MEP was recorded simultaneously in all patients who were treated with minimally invasive procedures, and the latency of MEP decreased compared with the MT group. As FA values of the CST in internal capsule increased and MEP appeared with its latency decreased, the modified National Institutes of Health Stroke Scale score decreased after the surgery. CONCLUSIONS: Minimally invasive procedures for thalamic hematoma evacuation could effectively reduce the degree of injury to the function as observed by a combination of DTI and MEP measurements.