Direct visualization of Agrobacterium‐delivered VirE2 in recipient cells

Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single‐stranded DNA molecules (T–DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split‐GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2‐GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1–10) was expressed in recipient cells. Upon delivery of VirE2‐GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2‐GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium‐delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf‐infiltration assay; most of VirE2 moved at a speed of 1.3–3.1 μm sec^?1 in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2‐GFP formed filamentous structures of different lengths, even in the absence of T‐DNA. As a non‐natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium‐delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T‐DNA transformation for a non‐natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non‐natural host recipient. The split‐GFP approach could enable the real‐time visualization of VirE2 trafficking inside recipient cells.     

FGF gene family characterization provides insights into its adaptive evolution in Carnivora

Fibroblast growth factors (FGFs) encoded by the FGF gene family can regulate development and physiology in animals. However, their evolutionary characteristics in Carnivora are largely unknown. In this study, we identified 660 sequences of three types of FGF genes from 30 unannotated genomes of Carnivora animals (before 7th May 2020), and the FGF genes from 52 Carnivora species were analyzed through the method of comparative genomics. Phylogenetic and selective pressure analyses were carried out based on the FGF genes of these 52 Carnivora species. The phylogenetic analysis results demonstrated that the FGF gene family was divided into 10 subfamilies and that FGF5 formed one clade rather than belonging to the subfamilies of FGF4 and FGF6. The evolutionary analysis results showed that the FGF genes were prominently subjected to purifying selection and were highly conserved in the process of Carnivora evolution. We also carried out phylogenetic comparative analyses, which indicated that the habitat was one of the factors that shaped the evolution of Carnivora FGF genes. The FGF1 and FGF6 genes were positively selected in the Carnivora animals, and positive selection signals were detected for the FGF19 gene in semiaquatic Carnivora animals. In summary, we clarified the phylogenetic and evolutionary characteristics of Carnivora FGF genes and provided valuable data for future studies on evolutionary characterization of Carnivora animals.     

A Functional Insertion/Deletion Polymorphism in the Proximal Promoter of CD3G Is Associated with Susceptibility for Hepatocellular Carcinoma in Chinese Population

Hepatocellular carcinoma (HCC) represents the most common primary malignancy of the liver with a worldwide increasing incidence. Although the risk factors for HCC are well characterized, the molecular mechanisms responsible for malignant transformation of hepatocytes are not well understood. In this study, a case-control study including 291 HCC patients and 294 healthy controls was conducted to investigate the association between HCC susceptibility and with a 4-bp insertion/deletion polymorphism (rs66465034) in the proximal promoter of CD3G. Logistic regression analysis showed that the heterozygote and the homozygote 4-bp ins/ins confer a significantly increased risk of HCC after controlling for other covariates (adjusted odds ratio [OR]=1.51, 95% confidence interval [C.I.] 1.01-2.27, p=0.040; OR=1.71, 95% C.I. 1.07-2.89, p=0.025, respectively). Carriage of the 4-bp insertion allele was associated with a greatly increased risk of developing the disease (OR=1.30, 95% C.I. 1.02-1.64, p=0.027). Moreover, hepatitis B virus (HBV) stratification analysis showed that the differences between cases and controls were more obvious in HBV-positive than in the HBV-negative population, suggesting a possible role of this polymorphism in the immune regulation during HBV infection. Further, luciferase-based transient transfection assays revealed that rs66465034 can affect promoter activity of CD3G, indicating its possible functional significance. Our data suggested that common genetic polymorphisms in CD3G may influence HCC risk in Chinese population. Considering the relative small sample size, replication in other populations with larger sample size and further functional analysis are required for fully understanding the roles of CD3G polymorphisms in predisposition for HCC.     

Inhibition of invariant chain processing, antigen-induced proliferative responses, and the development of collagen-induced arthritis and experimental autoimmune encephalomyelitis by a small molecule cysteine protease inhibitor

Members of the papain family of cysteine proteases (cathepsins) mediate late stage processing of MHC class II-bound invariant chain (Ii), enabling dissociation of Ii, and binding of antigenic peptide to class II molecules. Recognition of cell surface class II/Ag complexes by CD4(+) T cells then leads to T cell activation. Herein, we demonstrate that a pan-active cathepsin inhibitor, SB-331750, attenuated the processing of whole cell Ii p10 to CLIP by Raji cells, and DBA/1, SJL/J, and C57BL/6 splenocytes. In Raji cells and C57BL/6 splenocytes, SB-331750 inhibited class II-associated Ii processing and reduced surface class II/CLIP expression, whereas in SB-331750-treated DBA/1 and SJL/J splenocytes, class II-associated Ii processing intermediates were undetectable. Incubation of lymph node cells/splenocytes from collagen-primed DBA/1 mice and myelin basic protein-primed SJL/J mice with Ag in the presence of SB-331750 resulted in concentration-dependent inhibition of Ag-induced proliferation. In vivo administration of SB-331750 to DBA/1, SJL/J, and C57BL/6 mice inhibited splenocyte processing of whole cell Ii p10 to CLIP. Prophylactic administration of SB-331750 to collagen-immunized/boosted DBA/1 mice delayed the onset and reduced the severity of collagen-induced arthritis (CIA), and reduced paw tissue levels of IL-1beta and TNF-alpha. Similarly, treatment of myelin basic protein-primed SJL/J lymph node cells with SB-331750 delayed the onset and reduced the severity of adoptively transferred experimental autoimmune encephalomyelitis (EAE). Therapeutic administration of SB-331750 reduced the severity of mild/moderate CIA and EAE. These results indicate that pharmacological inhibition of cathepsins attenuates CIA and EAE, potentially via inhibition of Ii processing, and subsequent Ag-induced T cell activation.     

A Perovskite Nanorod as Bifunctional Electrocatalyst for Overall Water Splitting

The development of highly efficient and low‐cost electrocatalysts for oxygen evolution reaction (OER) and hydrogen evolution reaction (HER) is paramount for water splitting associated with the storage of clean and renewable energy. Here, this study reports its findings in the development of a nanostructured perovskite oxide as OER/HER bifunctional electrocatalyst for overall water splitting. Prepared by a facile electrospinning method, SrNb_0.1Co_0.7Fe_0.2O_3–δ perovskite nanorods (SNCF‐NRs) display excellent OER and HER activity and stability in an alkaline solution, benefiting from the catalytic nature of perovskites and unique structural features. More importantly, the SNCF‐NR delivers a current density of 10 mA cm^?2 at a cell voltage of merely ≈1.68 V while maintaining remarkable durability when used as both anodic and cathodic catalysts in an alkaline water electrolyzer. The performance of this bifunctional perovskite material is among the best ever reported for overall water splitting, offering a cost‐effective alternative to noble metal based electrocatalysts.     

Molecular characteristics of two new <Emphasis Type="Italic">waxy</Emphasis> mutations in China waxy maize

The waxy gene mutation causes waxy maize grain to have a sticky quality. China has numerous waxy maize landraces and is thought to be the place of origin of waxy maize. The most abundant waxy maize resources in China are located in the Yunnan province and its surrounding areas. We collected 57 waxy maize landraces from Yunnan province and cloned and sequenced the waxy gene from its fourth to eighth exon. Two new waxy gene mutations, named wx-Cin4 and wx-124, were identified. The wx-Cin4 mutation is a 466-bp retrotransposon inserted into exon six. The wx-124 mutation is a 116-bp miniature inverted-repeat transposable element inserted into exon seven. This is the first time a 124-type mutation has been found in a maize waxy gene. The discovery of the two specific waxy mutations from landraces collected in Yunnan province provides new evidence supporting the hypothesis that China is the origin area for waxy maize.