Genome editing in rice using CRISPR/Cas12i3.

The CRISPR/Cas type V-I is a family of programmable nuclease systems that prefers a T-rich protospacer adjacent motif (PAM) and is guided by a short crRNA. In this study, the genome-editing application of Cas12i3, a type V-I family endonuclease, was characterized in rice. We developed a CRIPSR/Cas12i3-based Multiplex direct repeats (DR)-spacer Array Genome Editing (iMAGE) system that was efficient in editing various genes in rice. Interestingly, iMAGE produced chromosomal structural variations with a higher frequency than CRISPR/Cas9. In addition, we developed base editors using deactivated Cas12i3 and generated herbicide-resistant rice plants using the base editors. These CRIPSR/Cas12i3-based genome editing systems will facilitate precision molecular breeding in plants.

An easily-performed high-throughput method for plant genomic DNA extraction.

Genomic DNA isolation is a crucial technique for researchers studying plant molecular biology. A current widely-used protocol for DNA extraction needs a pestle and mortal for each sample and consumes a large amount of liquid nitrogen in grinding the samples. Most high-throughput methods depend on expensive machines for tissue homogenization. Here we developed a CTAB-based DNA extraction method using 2.0 mL microcentrifuge tubes for sample processing. This protocol has the advantages that it is suitable for a variety of plants, easily-performed without special equipment, and high-throughput; it effectively avoids sample cross-contamination, and is inexpensive, rapid and safe.

Blockage of thymic stromal lymphopoietin signaling improves acute lung injury in mice by regulating pulmonary dendritic cells.

OBJECTIVES: To investigate the effects of blockage of thymic stromal lymphopoietin (TSLP) signaling by TSLP receptor (TSLPR)-immunoglobulin (Ig) on acute lung injury (ALI) induced by lipopolysaccharide (LPS). METHODS: C57BL/6 mice received TSLPR-Ig or controlled-Ig before being induced ALI. Lung wet/dry (W/D) weight ratio was recorded. Neutrophil number and albumin concentration of bronchoalveolar lavages fluids (BALF) were determined. Besides, bone marrow dendritic cells (BMDCs) were separated and cultured with medium, TSLP, TSLP plus TSLPR-Ig or TSLP plus controlled-Ig. Protein expression levels of TSLP in lung tissues, phosphorylation extracellular regulated protein kinases (pERK) 1/2, p38, and signal transducers and activators of transcription (STAT) 3 in BMDCs were analyzed using Western blotting. Expression of CD40, CD80 and CD86 on pulmonary DCs and BMDCs was determined using flow cytometry (FCM). RESULTS: The W/D ratio, neutrophil number and albumin concentration were significantly decreased in the TSLPR-Ig group compared with the controlled-Ig and model group. Moreover, there was a noticeable decrease in CD40, CD80 or CD86 expression by TSLPR-Ig on both pulmonary DCs and BMDCs. The protein levels of TSLP, pERK1 and STAT3 were significantly decreased by TSLPR-Ig. However, no significant differences were found in p38 and pERK2. CONCLUSION: These results suggest that TSLP may be involved in ALI, and blockage of TSLP signaling using TSLPR-Ig improves ALI at least in part by regulation of DCs functions. The underling downstream signaling mediated by TSLP might be associated with activating the ERK1 and STAT3 signaling pathway.

[Effect of shenmai injection on diaphragmatic fatigue in children with respiratory failure].

OBJECTIVE: To study the effect of Shenmai injection (SMI) on diaphragmatic fatigue in children with respiratory failure. METHODS: Thirty-five cases of children respiratory failure with diaphragmatic fatigue were divided into two groups. The control group was treated with comprehensive therapy including anti-infection, oxygen inhalation and parenteral nutrition, etc. The SMI group was treated with SMI intravenously, besides the comprehensive therapy as in the control group. Taking electrical impedance respirogram (IRG) as criterion of therapeutic effect, the effective cases after 30 min medication, time for diaphragmatic fatigue disappearance, as well as arterial blood gas analysis before and after treatment were analyzed. RESULTS: (1) In 30 min after medication, the effective cases in the SMI group (15/18) were more than that in the control group (4/17, P < 0.01); (2) Blood pH increased and PaCO2 decreased in both groups after treatment, but the decrease of PaCO2 was more significant in the SMI group (P < 0.05); (3) Time of diaphragmatic fatigue disappearance in the SMI group was shorter than that in the control group (P < 0.01). CONCLUSION: SMI is an effective drug for treatment of diaphragmatic fatigue in children with less adverse effect, and worthwhile for spreading in clinical practice.

[Diagnostic study on children's diaphragmatic fatigue].

OBJECTIVE: In the recent twenty years, the diaphragmatic contraction, relaxation functions and electric activity have been explored through electromyography (EMG) and transdiaphragmatic pressure (Pdi) determination. But these techniques required some complex and expensive instruments, so the diagnosis and treatment of children's diaphragmatic fatigue have not been well evaluated. The present study explored the diagnosis of children's diaphragmatic fatigue through measuring ribcage-abdomen motion and analyzed its asynchrony. METHODS: Fifty-three children (male 37, female 16, and age rage from 1 months to 9 years) with respiratory rate > 30 breaths/min, heart rate > 110 beats/min, and respiratory dysfunction had asynchronized ribcage-abdomen motion showed by impedance respirograph (IRG). The authors observed whether ribcage-abdomen motion was synchronic and calculated M levels (staggered peak time/total duration of the breathing cycle). The ribcage and abdomen outputs were displayed on vertical (for rib cage) and horizontal (for abdomen) axes of X-Y instrument. In addition, the change of respiratory frequency and heart rate was observed and arterial blood-gas analysis was also performed. RESULTS: (1) M levels in one-dimensional IRG were positively correlated with alpha angle in two-dimensional IRG (r = 0.956, P < 0.001). Asynchronized respiratory motions could be divided into three types. type I showed completely contra-directional movements of respiration, M levels for (48.1 +/- 4.4)%, an irregularly clockwise loop in the two dimensional IRG, and alpha angle for (138.3 +/- 15.0) degrees. In type II, one dimensional IRG showed displaced peak of the chest and abdomen motion curves, M levels were (16.5 +/- 4.7)%, two dimensional IRG was displaced in a counterclockwise direction, and alpha angle was (55.3 +/- 10.8) degrees. In type III, abdominal motion curve of one dimensional IRG had double peaks, M levels were 0, two dimensional IRG was presented as 8-shaped double circles, alpha angle was (41.3 +/- 3.8) degrees; (2) pH levels in the patients with type I and type II diaphragmatic fatigue were significantly lower, and PCO(2) levels were significantly higher than those with type III or in the normal subjects (P < 0.001 for all), but there was no statistically significant difference between type III and the normal subjects (P > 0.05); (3) Both of respiratory rate and heart rate in type I, type II and type III were higher than those in the normal subjects (all P < 0.001), and the differences among the three types were significant (P < 0.001 for all); (4) Both M levels and alpha angle were negatively correlated with pH levels (r = -0.514, P < 0.001 and r = -0.497, P < 0.001), while positively correlated with PCO(2) levels (r = 0.672, P < 0.001 and r = 0.625, P = 0.01). CONCLUSIONS: (1) IRG can be reliably used to diagnose children's diaphragmatic fatigue. This technique is simple and easy to perform and non-invasive. It is therefore worthy of recommending for further clinical investigations. (2) According to the characteristics of IRG, diaphragmatic fatigue can be divided into three types. (3) The development of children's diaphragmatic fatigue has a series of characteristic changes. (4) To avoid the patients suffering from respiratory failure, it is the key time to adopt the policies of prevention and treatment when IRG shows signs of type III diaphragmatic fatigue.