The use of SARS-CoV-2-related coronaviruses from bats and pangolins to polarize mutations in SARS-Cov-2

正Dear Editor,The coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 coronavirus has become a global pandemic.The SARS-CoV-2 genome has a similarity of 96.2%to that of RaTG13, a bat SARS-CoV-2-related coronavirus detected in Rhinolophus affinis (Paraskevis et al., 2020; Zhou et al.,2020). The SARS-CoV-2 genome also has 85.5%-92.4%     

Efficacy of RyR2 inhibitor EL20 in induced pluripotent stem cell-derived cardiomyocytes from a patient with catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac arrhythmia syndrome that often leads to sudden cardiac death. The most common form of CPVT is caused by autosomal-dominant variants in the cardiac ryanodine receptor type-2 (RYR2) gene. Mutations in RYR2 promote calcium (Ca²⁺) leak from the sarcoplasmic reticulum (SR), triggering lethal arrhythmias. Recently, it was demonstrated that tetracaine derivative EL20 specifically inhibits mutant RyR2, normalizes Ca²⁺ handling and suppresses arrhythmias in a CPVT mouse model. The objective of this study was to determine whether EL20 normalizes SR Ca²⁺ handling and arrhythmic events in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a CPVT patient. Blood samples from a child carrying RyR2 variant RyR2 variant Arg-176-Glu (R176Q) and a mutation-negative relative were reprogrammed into iPSCs using a Sendai virus system. iPSC-CMs were derived using the Stemdiff^TM kit. Confocal Ca²⁺ imaging was used to quantify RyR2 activity in the absence and presence of EL20. iPSC-CMs harbouring the R176Q variant demonstrated spontaneous SR Ca²⁺ release events, whereas administration of EL20 diminished these abnormal events at low nanomolar concentrations (IC₅₀ = 82 nM). Importantly, treatment with EL20 did not have any adverse effects on systolic Ca²⁺ handling in control iPSC-CMs. Our results show for the first time that tetracaine derivative EL20 normalized SR Ca²⁺ handling and suppresses arrhythmogenic activity in iPSC-CMs derived from a CPVT patient. Hence, this study confirms that this RyR2-inhibitor represents a promising therapeutic candidate for treatment of CPVT.     

Expression profiles and potential roles of transfer RNA-derived small RNAs in atherosclerosis

Knowledge regarding the relationship between the molecular mechanisms underlying atherosclerosis (AS) and transfer RNA-derived small RNAs (tsRNAs) is limited. This study illustrated the expression profile of tsRNAs, thus exploring its roles in AS pathogenesis. Small RNA sequencing was performed with four atherosclerotic arterial and four healthy subject samples. Using bioinformatics, the protein-protein interaction network and cellular experiments were constructed to predict the enriched signalling pathways and regulatory roles of tsRNAs in AS. Of the total 315 tsRNAs identified to be dysregulated in the AS group, 131 and 184 were up-regulated and down-regulated, respectively. Interestingly, the pathway of the differentiated expression of tsRNAs in cell adhesion molecules (CAMs) was implicated to be closely associated with AS. Particularly, tRF-Gly-GCC might participate in AS pathogenesis via regulating cell adhesion, proliferation, migration and phenotypic transformation in HUVECs and VSMCs. In conclusion, tsRNAs might help understand the molecular mechanisms of AS better. tRF-Gly-GCC may be a promising target for suppressing abnormal vessels functions, suggesting a novel strategy for preventing the progression of atherosclerosis.     

Arabidopsis Villins Promote Actin Turnover at Pollen Tube Tips and Facilitate the Construction of Actin Collars

Apical actin filaments are crucial for pollen tube tip growth. However, the specific dynamic changes and regulatory mechanisms associated with actin filaments in the apical region remain largely unknown. Here, we have investigated the quantitative dynamic parameters that underlie actin filament growth and disappearance in the apical regions of pollen tubes and identified villin as the major player that drives rapid turnover of actin filaments in this region. Downregulation of Arabidopsis thaliana VILLIN2 (VLN2) and VLN5 led to accumulation of actin filaments at the pollen tube apex. Careful analysis of single filament dynamics showed that the severing frequency significantly decreased, and the lifetime significantly increased in vln2 vln5 pollen tubes. These results indicate that villin-mediated severing is critical for turnover and departure of actin filaments originating in the apical region. Consequently, the construction of actin collars was affected in vln2 vln5 pollen tubes. In addition to the decrease in severing frequency, actin filaments also became wavy and buckled in the apical cytoplasm of vln2 vln5 pollen tubes. These results suggest that villin confers rigidity upon actin filaments. Furthermore, an observed decrease in skewness of actin filaments in the subapical region of vln2 vln5 pollen tubes suggests that villin-mediated bundling activity may also play a role in the construction of actin collars. Thus, our data suggest that villins promote actin turnover at pollen tube tips and facilitate the construction of actin collars.     

Dickkopf-1 Is Oncogenic and Involved in Invasive Growth in Non Small Cell Lung Cancer

Dickkopf-1 (DKK1) is an inhibitor of the Wnt/β-catenin signaling pathway. However, the role of DKK1 in the progression of non small cell lung cancer (NSCLC) is not fully understood. In this study, RT-PCR and Western blot were used to examine the expression of DKK1 in a panel of ten human NSCLC cell lines and NSCLC tissues. DKK1 expression was highly transactivated in the great majority of these cancer lines. The expression of DKK1 was upregulated on both mRNA and protein levels in NSCLC tissues compared with the adjacent normal lung tissues. Immunohistochemistry and immunofluoresence revealed that DKK1 was mainly distributed in the cytoplasm in both carcinoma tissues and cell lines. DKK1 protein expression was also evaluated in paraffin sections from 102 patients with NSCLC by immunohistochemistry, and 65(63.73%)tumors were DKK1 positive. Relative analysis showed a significant relationship between DKK1 positive expression and lymph node metastasis(P<0.05). Patients with DKK1-positive tumors had poorer DFS than those with negative ESCC (5-year DFS; 15.4% versus 27%, P = 0.007). To further explore the biological effects of DKK1 in NSCLC cells, we over-expressed DKK1 in NSCLC 95C cell using eukaryotic expression vector pCMV-Tab-2b and performed a knockdown of DKK1 in LTEP-a-2 cell using a short hairpin RNA expression vector pSilencer 5.1. DKK1 did not have any effect on proliferation, but seemed to play a role in migration and invasion capability. Overexpression of DKK1 promotes migratory and invasive activity of 95C, while DKK1 knockdown resulted in the suppression of migration and invasion potentials of LTEP-a-2 cell. Taken together, these results indicate that DKK1 may be a crucial regulator in the progression of NSCLC. DKK1 might be a potential therapeutic target in NSCLC.     

Overexpression of an Acidic Endo-β-1,3-1,4-glucanase in Transgenic Maize Seed for Direct Utilization in Animal Feed

Background

Incorporation of exogenous glucanase into animal feed is common practice to remove glucan, one of the anti-nutritional factors, for efficient nutrition absorption. The acidic endo-β-1,3-1,4-glucanase (Bgl7A) from Bispora sp. MEY-1 has excellent properties and represents a potential enzyme supplement to animal feed.

Methodology/Principal Findings

Here we successfully developed a transgenic maize producing a high level of Bgl7AM (codon modified Bgl7A) by constructing a recombinant vector driven by the embryo-specific promoter ZM-leg1A. Southern and Western blot analysis indicated the stable integration and specific expression of the transgene in maize seeds over four generations. The β-glucanase activity of the transgenic maize seeds reached up to 779,800 U/kg, about 236-fold higher than that of non-transgenic maize. The β-glucanase derived from the transgenic maize seeds had an optimal pH of 4.0 and was stable at pH 1.0–8.0, which is in agreement with the normal environment of digestive tract.

Conclusion/Significance

Our study offers a transgenic maize line that could be directly used in animal feed without any glucanase production, purification and supplementation, consequently simplifying the feed enzyme processing procedure.     

Soil cadmium toxicity and nitrogen deposition differently affect growth and physiology in <Emphasis Type="Italic">Toxicodendron vernicifluum</Emphasis> seedlings

To ultimately determine whether different levels of soil nitrogen (N) deposition can modify the detrimental effects of cadmium (Cd), the seedlings of Toxicodendron vernicifluum (Strokes) F. A. Barkley were exposed to soil Cd stress (0, 5 and 15 mg kg^?1 dry soil), N deposition (0, 13 and 40 mg kg^?1 dry soil) and their combinations. Soil Cd stress caused damage in plant growth, photosynthesis and other physiological indexes, and in the ultrastructure of mesophyll cells. The effects of N deposition on growth, lipid peroxidation and enzyme activities depended on the relative amounts of N supplied. The combination of low N deposition and Cd stress was positive to plant growth, photosynthesis and enzyme activities, and it caused lower levels of Cd accumulation and lipid peroxidation compared with the effect of Cd stress alone. The combination of high N deposition and Cd stress led to a higher Cd accumulation and lipid peroxidation, and to lower enzyme activities, as compared with the effect of Cd stress alone. T. vernicifluum was found to be sensitive to soil Cd stress. Soil Cd had detrimental effects on T. vernicifluum seedlings, but the tolerance of T. vernicifluum to Cd increased under low N deposition.     

Development of a high-efficient transformation system of Bacillus pumilus strain DX01 to facilitate gene isolation via gfp-tagged insertional mutagenesis and visualize bacterial colonization of rice roots

A Tn5 transposition vector, pMOD-tet-egfp, was constructed and used for the random insertional mutagenesis of Bacillus pumilus. Various parameters were investigated to increase the transformation efficiency B. pumilus DX01 via Tn5 transposition complexes (transposome): bacterial growth phase, type of electroporation buffer, electric field strength, and recovery medium. Transformation efficiency was up to 3?×?10⁴?transformants/μg of DNA under the optimized electroporation conditions, and a total of 1,467 gfp-tagged transformants were obtained. Fluorescence-activated cell sorting analysis showed that all gfp-tagged bacterial cells expressed GFP, indicating that foreign DNA has been successfully integrated into the genome of B. pumilus and expressed. Finally, flanking DNA sequences were isolated from several transformants and colonization of rice roots by B. pumilus DX01 was also studied. The method developed here will be useful for creating an insertion mutant library of gram-positive bacteria, thus facilitating their molecular genetic and cytological studies.     

Altitudinal variation in growth, photosynthetic capacity and water use efficiency of Abies faxoniana Rehd. et Wils. seedlings as revealed by reciprocal transplantations

Altitudinal variation in morphological, physiological and biochemical characteristics between two provenances of Abies faxoniana Rehd. et Wils. from contrasting elevations (3,500 and 2,850 m) was investigated by reciprocal transplantations in the eastern Qinghai-Tibetan Plateau. From each altitude, 54 seedlings were selected, of which 27 seedlings were simply transplanted at their original altitude, while the other 27 seedlings were reciprocally transplanted to another altitude. The results showed that there is evident altitudinal variation in growth, photosynthetic capacity and water use efficiency among A. faxoniana seedlings. Transplantation of seedlings to a lower altitude decreased the leaf pigment content, leaf N content and water use efficiency, but increased CE, P _n, LMR and PNUE, consequently promoting NAR, the biomass accumulation and branchlet length increment. Besides, the increase in the C/N ratio reduced the risk of pathogen attack. Based on the substantial plasticity in the responses of the seedlings, we infer that the ongoing climate warming might facilitate the growth of A. faxoniana seedlings at high altitudes.