The revolution of the biology of the genome

Sequence data of entire eukaryotic genomes and their detailed comparison have provided new evidence on genome evolution. The major mechanisms involved in the increase of genome sizes are polyploidization and gene duplication.Subsequent gene silencing or mutations, preferentially in regulatory sequences of genes, modify the genome and permit the development of genes with new properties. Mechanisms such as lateral gene transfer, exon shuffling or the creation of new genes by transposition contribute to the evolution of a genome, but remain of relatively restricted relevance.Mechanisms to decrease genome sizes and, in particular, to remove specific DNA sequences, such as blocks of satellite DNAs, appear to involve the action of RNA interference (RNAi). RNAi mechanisms have been proven to be involved in chromatin packaging related with gene inactivation as well as in DNA excision during the macronucleus development in ciliates.     

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance （DDT） has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen （PCNA） by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis （TLS） with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer.     

Two distinct conjugal transfer systems on Streptomyces plasmid pZL1

The mechanism of conjugal transfer of plasmids in Gram-negative and unicellular Gram-positive bacteria is commonly via a type IV secretion system （T4SS） [1]. The genes encoding the T4S proteins are usually arranged in a single operon or a few operons. In Gram-negative Agrobacterium tumefaciens, the T4SS is encoded by the virB and virD operons （11 and 5 genes, respectively） [2]. Streptomyces are multicellular mycelial Gram-positive bac- teria that form unicellular spores. There are fundamental dif- ferences in the mechanisms of conjugal transfer between Streptomyces plasmids and those of Gram-negative and uni- cellular Gram-positive bacteria. Conjugal transfer of Streptomyces plasmids requires a tra gene encoding an FtsK/SpolIIE-family DNA translocator and a few adjacent genes [3]. No bacterial T4SS has previously been found on Streptomyces conjugative plasmids. We reported here the co- existence of both a T4SS-like and an FtsK/SpolIIE-family DNA translocator on a 128-kb Streptomyces plasmid, pZL 1.     

Mucosal-associated invariant T cells from induced pluripotent stem cells:A novel approach for modeling human diseases

Mice have frequently been used to model human diseases involving immune dysregulation such as autoimmune and inflammatory diseases.These models help elucidatethe mechanisms underlying the disease and in the development of novel therapies.However,if mice are deficient in certain cells and/or effectors associated with human diseases,how can their functions be investigated in this species?Mucosal-associated invariant T(MAIT)cells,a novel innate-like T cell family member,are a good example.MAIT cells are abundant in humans but scarce in laboratory mice.MAIT cells harbor an invariant T cell receptor and recognize nonpeptidic antigens vitamin B2metabolites from bacteria and yeasts.Recent studies have shown that MAIT cells play a pivotal role in human diseases such as bacterial infections and autoimmune and inflammatory diseases.MAIT cells possess granulysin,a human-specific effector molecule,but granulysin and its homologue are absent in mice.Furthermore,MAIT cells show poor proliferation in vitro.To overcome these problems and further our knowledge of MAIT cells,we have established a method to expand MAIT cells via induced pluripotent stem cells(iP SCs).In this review,we describe recent advances in the field of MAIT cell research and our approach for human disease modeling with iP SCderived MAIT cells.     

Detection of clustered DNA lesions: Biological and clinical applications

Humans are daily exposed to background radiation and various sources of oxidative stress. My research has focused in the last 12 years on the effects of ionizing radiation on DNA, which is considered as the key target of radiation in the cell. Ionizing radiation and endogenous cellular oxidative stress can also induce closely spaced oxidatively induced DNA lesions called "clusters" of DNA damage or locally multiply damage sites, as first introduced by John Ward. I am now interested in the repair mechanisms of clustered DNA damage, which is considered as the most difficult for the cell to repair. A main part of my research is devoted to evaluating the role of clustered DNA damage in the promotion of carcinogenesis in vitro and in vivo . Currently in my laboratory, there are two main ongoing projects. (1) Study of the role of BRCA1 and DNA-dependent protein kinase catalytic subunit repair proteins in the processing of clustered DNA damage in human cancer cells. For this project, we use several tumor cell lines, such as breast cancer cell lines MCF-7 and HCC1937 (BRCA1 deficient) and human glioblastoma cells MO59J/K; and (2) Possible use of DNA damage clusters as novel cancer biomarkers for prognostic and therapeutic applications related to modulation of oxidative stress. In this project human tumor and mice tissues are being used.     

Epigenetic integrity of paternal imprints enhances the developmental potential of androgenetic haploid embryonic stem cells

The use of two inhibitors of Mek1/2 and Gsk3β(2i)promotes the generation of mouse diploid and haploid embryonic stem cells(ESCs)from the inner cell mass of biparental and uniparental blastocysts,respectively.However,a system enabling long-term maintenance of imprints in ESCs has proven challenging.Here,we report that the use of a two-step a2i(alternative two inhibitors of Src and Gsk3β,TSa2i)derivation/culture protocol results in the establishment of androgenetic haploid ESCs(AG-haESCs)with stable DNA methylation at paternal DMRs(differentially DNA methylated regions)up to passage 60 that can efficiently support generating mice upon oocyte injection.We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations.Furthermore,we demonstrate that TSa2itreated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation.Strikingly,AGhaESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells,in part through the enhanced proliferation of H19-DMR hypermethylated cells.Together,we establish AG-haESCs that can longterm maintain paternal imprints.     

The host type I interferon response to viral and bacterial infections

Type I interferons (IFN) are well studied cytokines with anti-viral and immune-modulating functions. Type I IFNs are produced following viral infections, but until recently, the mechanisms of viral recognition leading to IFN production were largely unknown. Toll like receptors (TLRs) have emerged as key transducers of type I IFN during viral infections by recognizing various viral components. Furthermore, much progress has been made in defining the signaling pathways downstream of TLRs for type I IFN production. TLR7 and TLR9 have become apparent as universally important in inducing type I IFN during infection with most viruses, particularly by plasmacytoid dendritic cells. New intracellular viral pattern recognition receptors leading to type I IFN production have been identified. Many bacteria can also induce the up-regulation of these cytokines. Interestingly, recent studies have found a detrimental effect on host cells if type Ⅰ IFN is produced during infection with the intracellular gram-positive bacterial pathogen, Listeria monocytogenes. This review will discuss the recent advances made in defining the signaling pathways leading to type I IFN production.     

Activation of p53 in gastric cancer cells by tripchlorolide specifically induces apoptosis

There are two possible outcomes when DNA damage occurs in normal mammalian cells: either induction of cell-cycle checkpoint which inhibits the progress of the cell cycles as well as activates DNA repair pathways, or activation of apoptosis to eliminate damaged cells. The p53 tumour-suppressor gene plays a key role in selecting these pathways. In our present works, the human gastric cancer cell line AGS was treated with tripchlorolide, a potent antitumor compound purified from a Chinese herb Tripterygium Wilfordii Hook. Single cell gel electrophoresis (Comet assay) showed that the treatment of tripchlorolide resulted in DNA damage in AGS cells. The damaged AGS cells went through apoptosis, which was time- and dose- dependent.     

Arabidopsis Profilin Isoforms,PRF1 and PRF2 Show Distinctive Binding Activities and Subcellular Distributions

Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fuorescent protein （GFP） tag and expressed in Escherichia coil and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgenic A. thaliana lines revealed that 35S：：GFP-PRF1 formed a filamentous network, while 35S：：GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profllin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive Iocalizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.