Transfer Genes Research Articles

Determination and Analysis of the Putative AcaCD-Responsive Promoters of Salmonella Genomic Island 1.

The integrative genomic island SGI1 and its variants confer multidrug resistance in numerous Salmonella enterica serovariants and several Proteus mirabilis and Acinetobacter strains. SGI1 is mobilized by the IncA/C family plasmids. The island exploits not only the conjugation apparatus of the plasmid, but also utilizes the plasmid-encoded master regulator AcaCD to induce the excision and formation of its transfer-competent form, which is a key step in the horizontal transfer of SGI1. Triggering of SGI1 excision occurs via the AcaCD-dependent activation of xis gene expression. AcaCD binds in Pxis to an unusually long recognition sequence. Beside the Pxis promoter, upstream regions of four additional SGI1 genes, S004, S005, S012 and S018, also contain putative AcaCD-binding sites. Furthermore, SGI1 also encodes an AcaCD-related activator, FlhDCSGI1, which has no known function. Here, we have analysed the functionality of the putative AcaCD-dependent promoter regions and proved their activation by either AcaCD or FlhDCSGI1. Moreover, we provide evidence that both activators act on the same binding site in Pxis and that FlhDCSGI1 is able to complement the acaCD deletion of the IncA/C family plasmid R16a. We determined the transcription start sites for the AcaCD-responsive promoters and showed that orf S004 is expressed probably from a different start codon than predicted earlier. Additionally, expression of S003 from promoter PS004 was ruled out. Pxis and the four SGI1 promoters examined here also lack obvious -35 promoter box and their promoter profile is consistent with the class II-type activation pathway. Although the role of the four additionally analysed AcaCD/FlhDCSGI1-controlled genes in transfer and/or maintenance of SGI1 is not yet clear, the conservation of the whole region suggests the existence of some selection for their functionality.

A new high-throughput sequencing method for determining diversity and similarity of T cell receptor (TCR) α and β repertoires and identifying potential new invariant TCR α chains.

BackgroundHigh-throughput sequencing of T cell receptor (TCR) genes is a powerful tool for analyses of antigen specificity, clonality and diversity of T lymphocytes. Here, we developed a new TCR repertoire analysis method using 454 DNA sequencing technology in combination with an adaptor-ligation mediated polymerase chain reaction (PCR). This method allows the amplification of all TCR genes without PCR bias. To compare gene usage, diversity and similarity of expressed TCR repertoires among individuals, we conducted next-generation sequencing (NGS) of TRA and TRB genes in peripheral blood mononuclear cells from 20 healthy human individuals.ResultsFrom a total of 267,037 sequence reads from 20 individuals, 149,216 unique sequence reads were identified. Preferential usage of several V and J genes were observed while some recombinations of TRAV with TRAJ appeared to be restricted. The extent of TCR diversity was not significantly different between TRA and TRB, while TRA repertoires were more similar between individuals than TRB repertoires were. The interindividual similarity of TRA depended largely on the frequent presence of shared TCRs among two or more individuals. A publicly available TRA had a near-germline TCR with a shorter CDR3. Notably, shared TRA sequences, especially those shared among a large number of individuals’, often contained TCRα related with invariant TCRα derived from invariant natural killer T cells and mucosal-associated invariant T cells.ConclusionThese results suggest that retrieval of shared TCRs by NGS would be useful for the identification of potential new invariant TCRα chains. This NGS method will enable the comprehensive quantitative analysis of TCR repertoires at a clonal level.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-016-0177-5) contains supplementary material, which is available to authorized users.

OS2.6 Identification of IDH1R132H-specific T cell receptors from a humanized mouse model and from glioma patients treated with an IDH1R132H-specific peptide vaccine

AbstractCentral to clinically relevant immunotherapy of gliomas are the discovery of tumor-specific antigens and the development of therapeutic strategies that effectively exploit them. One such example is the mutation in the gene encoding for isocitrate dehydrogenase 1 (IDH1). The point mutation IDH1R132H is the most frequent one in grade II and grade III gliomas and secondary glioblastomas. We have previously shown that a peptide vaccine encoding the IDH1R132H epitope evokes an MHC II restricted CD4+ T Helper-1 cell (TH1) immune response and is therapeutic in an MHC-humanized A2.DR1 mouse tumor model.To determine the clonality in the T-cell receptor (TCR) repertoire of the IDH1R132H specific T-cells, a T-cell line was developed from splenocytes of vaccinated A2.DR1 mice by re-stimulations with autologous, naive irradiated and p132–142 (IDH1R132H)-loaded splenocytes. The cells were then sorted for IDH1R132H specificity by tetramer staining. Deep sequencing analyses of the variable V-J or V-D-J regions of the TRA and TRB genes, which code for the hyper-variable regions of the TCR-alpha and TCR-beta chains, respectively, revealed multiple dominant TRA and TRB sequences. Full length TCRs were constructed by pairing the two most dominant TRA and TRB sequences and cloned into expression vectors for functional validation.In addition to identifying the murine HLA-DR1-restricted TCR, we have analysed IDH1R132H-specific TCRs from a patient with a progressive IDH1R132H-mutated astrocytoma, who has been treated with an IDH1(R132H) vaccine on a compassionate use basis. Single T-cells from this patient, who had a high baseline mutation-specific T-cell response, specifically responding to IDH1(R132H), were expanded in vitro and subjected to single-cell-sorting and subsequent TCR sequencing. 11 different TCR sequences have been identified.The long-term goal is to develop an adoptive T-cell therapy using autologous IDH1R132H-specific T cells.

MPS 08-05 Association of CETP gene D442G polymorphisms with myocardial infarction and stroke in Chinese population

Objective: Genetic variants of proteins involved in lipid metabolism may play an important role in determining the susceptibility for complications associated with cardiovascular and cerebrovascular diseases. Goal of the present study was to determine the association of cholesteryl ester transfer (CETP) gene D442G (rs2303790) polymorphisms with myocardial infarction and stroke. Design and Method: We conducted two case–control association studies (one cohort of 2325 myocardial infarction patients and 2620 unrelated controls, another cohort of 3960 stroke patients and 3654 controls)from the Chinese population. Genotyping of the rs2303790 SNP was performed by the TaqMan Real Time PCR method. Results: In both cohorts CETP D442G (rs2303790) polymorphisms was associated with levels of HDL cholesterol and ApoA1 (all p < 0.001). After adjusting for age, sex, BMI and other factors, CETP D442G has no association with myocardial infarction and hemorrhagic stroke. In subgroup of stroke, the SNP showed a significant association with Ischemic stroke adjusting by HDL cholesterol (odds ratio 1.39, 95%CI: 1.098–1.772, p = 0.0065, dominant model) or ApoA1 (odds ratio 1.47, 95%CI: 1.152–1.876, p = 0.0024, dominant model). Conclusions: The present study has shown that in the Chinese population CETP D442G polymorphisms was associated with HDL cholesterol and ApoA1 levels, but not myocardial infarction and hemorrhagic stroke. The detailed physiological effects of SNP and Ischemic stroke needed to be future defined.

Effect of octylphenol and bisphenol A on cation transfer genes in mouse placenta during pregnancy

Effect of octylphenol and bisphenol A on cation transfer genes in mouse placenta during pregnancy

Contribution of Multiple Inter-Kingdom Horizontal Gene Transfers to Evolution and Adaptation of Amphibian-Killing Chytrid, Batrachochytrium dendrobatidis.

Amphibian populations are experiencing catastrophic declines driven by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Although horizontal gene transfer (HGT) facilitates the evolution and adaptation in many fungi by conferring novel function genes to the recipient fungi, inter-kingdom HGT in Bd remains largely unexplored. In this study, our investigation detects 19 bacterial genes transferred to Bd, including metallo-beta-lactamase and arsenate reductase that play important roles in the resistance to antibiotics and arsenates. Moreover, three probable HGT gene families in Bd are from plants and one gene family coding the ankyrin repeat-containing protein appears to come from oomycetes. The observed multi-copy gene families associated with HGT are probably due to the independent transfer events or gene duplications. Five HGT genes with extracellular locations may relate to infection, and some other genes may participate in a variety of metabolic pathways, and in doing so add important metabolic traits to the recipient. The evolutionary analysis indicates that all the transferred genes evolved under purifying selection, suggesting that their functions in Bd are similar to those of the donors. Collectively, our results indicate that HGT from diverse donors may be an important evolutionary driver of Bd, and improve its adaptations for infecting and colonizing host amphibians.

Folate deprivation induces cell cycle arrest at G0/G1 phase and apoptosis in hippocampal neuron cells through down-regulation of IGF-1 signaling pathway

Folate deficiency contributes to impaired adult hippocampal neurogenesis, yet the mechanisms remain unclear. Here we use HT-22 hippocampal neuron cells as model to investigate the effect of folate deprivation (FD) on cell proliferation and apoptosis, and to elucidate the underlying mechanism. FD caused cell cycle arrest at G0/G1 phase and increased the rate of apoptosis, which was associated with disrupted expression of folate transport and methyl transfer genes. FOLR1 and SLC46A1 were (P<0.01) down-regulated, while SLC19A1 was up-regulated (P<0.01) in FD group. FD cells exhibited significantly (P<0.05) higher protein content of BHMT, MAT2b and DNMT3a, as well as increased SAM/SAH concentrations and global DNA hypermethylation. The expression of the total and all the 3 classes of IGF-1 mRNA variants was significantly (P<0.01) down-regulated and IGF-1 concentration was decreased (P<0.05) in the culture media. IGF-1 signaling pathway was also compromised with diminished activation (P<0.05) of STAT3, AKT and mTOR. CpG hypermethylation was detected in the promoter regions of IGF-1 and FOLR1 genes, while higher SLC19A1 mRNA corresponded to hypomethylation of its promoter. IGF-1 supplementation in FD media significantly abolished FD-induced decrease in cell viability. However, IGF-1 had limited effect in rescuing the cell phenotype when added 24h after FD. Taken together, down-regulation of IGF-1 expression and signaling is involved in FD-induced cell cycle arrest and apoptosis in HT-22 hippocampal neuron cells, which is associated with an abnormal activation of methyl transfer pathway and hypermethylation of IGF-1 gene promoter.

Transcriptome analysis revealed that a quorum sensing system regulates the transfer of the pAt megaplasmid in Agrobacterium tumefaciens

BackgroundAgrobacterium tumefaciens strain P4 is atypical, as the strain is not pathogenic and produces a for this species unusual quorum sensing signal, identified as N-(3-hydroxy-octanoyl)-homoserine lactone (3OH,C8-HSL).ResultsBy sequence analysis and cloning, a functional luxI-like gene, named cinI, has been identified on the At plasmid of A. tumefaciens strain P4. Insertion mutagenesis in the cinI gene and transcriptome analyses permitted the identification of 32 cinI-regulated genes in this strain, most of them encoding proteins responsible for the conjugative transfer of pAtP4. Among these genes were the avhB genes that encode a type 4 secretion system (T4SS) involved in the formation of the conjugation apparatus, the tra genes that encode the DNA transfer and replication (Dtr) machinery and cinI and two luxR orthologs. These last two genes, cinR and cinX, exhibit an unusual organization, with the cinI gene surrounded by the two luxR orthologs. Conjugation experiments confirmed that the conjugative transfer of pAtP4 is regulated by 3OH,C8-HSL. Root colonization experiments indicated that the quorum sensing regulation of the conjugation of the pAtP4 does not confer a gain or a loss of fitness to the bacterial host in the tomato plant rhizosphere.ConclusionThis work is the first identification of the occurrence of a quorum sensing regulation of the pAt conjugation phenomenon in Agrobacterium.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3007-5) contains supplementary material, which is available to authorized users.

Viral and Synthetic RNA Vector Technologies and Applications.

Use of RNA is an increasingly popular method to transiently deliver genetic information for cell manipulation in basic research and clinical therapy. In these settings, viral and nonviral RNA platforms are employed for delivery of small interfering RNA and protein-coding mRNA. Technological advances allowing RNA modification for increased stability, improved translation and reduced immunogenicity have led to increased use of nonviral synthetic RNA, which is delivered in naked form or upon formulation. Alternatively, highly efficient viral entry pathways are exploited to transfer genes of interest as RNA incorporated into viral particles. Current viral RNA transfer technologies are derived from Retroviruses, nonsegmented negative-strand RNA viruses or positive-stranded Alpha- and Flaviviruses. In retroviral particles, the genes of interest can either be incorporated directly into the viral RNA genome or as nonviral RNA. Nonsegmented negative-strand virus-, Alpha- and Flavivirus-derived vectors support prolonged expression windows through replication of viral RNA encoding genes of interest. Mixed technologies combining viral and nonviral components are also available. RNA transfer is ideal for all settings that do not require permanent transgene expression and excludes potentially detrimental DNA integration into the target cell genome. Thus, RNA-based technologies are successfully applied for reprogramming, transdifferentiation, gene editing, vaccination, tumor therapy, and gene therapy.

SXT/R391 Integrative and Conjugative Elements (ICEs) Encode a Novel 'Trap-Door' Strategy for Mobile Element Escape.

Integrative conjugative elements (ICEs) are a class of bacterial mobile elements that have the ability to mediate their own integration, excision, and transfer from one host genome to another by a mechanism of site-specific recombination, self-circularisation, and conjugative transfer. Members of the SXT/R391 ICE family of enterobacterial mobile genetic elements display an unusual UV-inducible sensitization function which results in stress induced killing of bacterial cells harboring the ICE. This sensitization has been shown to be associated with a stress induced overexpression of a mobile element encoded conjugative transfer gene, orf43, a traV homolog. This results in cell lysis and release of a circular form of the ICE. Induction of this novel system may allow transfer of an ICE, enhancing its survival potential under conditions not conducive to conjugative transfer.

Low temperature acclimation with electrical stimulation enhance the biocathode functioning stability for antibiotics detoxification

Improvement of the stability of functional microbial communities in wastewater treatment system is critical to accelerate pollutants detoxification in cold regions. Although biocathode communities could accelerate environmental pollutants degradation, how to acclimate the cold stress and to improve the catalytic stability of functional microbial communities are remain poorly understood. Here we investigated the structural and functional responses of antibiotic chloramphenicol (CAP) reducing biocathode communities to constant low temperature 10 °C (10-biocathode) and temperature elevation from 10 °C to 25 °C (S25-biocathode). Our results indicated that the low temperature acclimation with electrical stimulation obviously enhanced the CAP nitro group reduction efficiency when comparing the aromatic amine product AMCl2 formation efficiency with the 10-biocathode and S25-biocathode under the opened and closed circuit conditions. The 10-biocathode generated comparative AMCl maximum as the S25-biocathode but showed significant lower dehalogenation rate of AMCl2 to AMCl. The continuous low temperature and temperature elevation both enriched core functional community in the 10-biocathode and S25-biocathode, respectively. The 10-biocathode functioning stability maintained mainly through selectively enriching cold-adapted functional species, coexisting metabolically similar nitroaromatics reducers and maintaining the relative abundance of key electrons transfer genes. This study provides new insights into biocathode functioning stability for accelerating environmental pollutants degradation in cold wastewater system.

252. Using Cre-Dependent In Vivo Selection to Identify AAV Variants That Enable Efficient and Widespread Gene Transfer to the Adult Central Nervous System

Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting the development of novel AAV capsids with more desirable transduction characteristics. We have developed a capsid selection method, called Cre-recombination-based AAV targeted evolution (CREATE), that enables the identification of AAV capsids that more efficiently transduce defined cell populations in vivo (Deverman et al. in press, Nature Biotechnology). We generated AAV capsid libraries and used CREATE to identify variants that cross the blood brain barrier and efficiently and widely transduce astrocytes in the mouse central nervous system (CNS) after intravenous injection. One variant, AAVPHP. B, transfers genes throughout the adult CNS with an efficiency that is 40- to 92-fold greater (depending on the CNS region) than that of the current standard, AAV9. It transduces the majority of astrocytes and neurons across multiple CNS regions, and in vitro, it transduces human neurons and astrocytes more efficiently than does AAV9. We are now evolving AAV-PHP.B for even greater transduction of specific CNS cell types as a means to both develop more effective vectors and to gain insight into the mechanism of enhanced transduction. Our identification of AAV-PHP.B and several other enhanced vectors after only two rounds of selection establishes CREATE as a powerful method to customize AAV vectors for biomedical applications.

Cytogenetic Behavior of Trigeneric Hybrid Progeny Involving Wheat, Rye and Psathyrostachys huashanica

Trigeneric hybrids are commonly used as bridges to transfer genes from some wild species to cultivated wheat and to measure the genomic interaction between donor species. We previously reported that trigeneric germplasms were produced by crossing wheat-Psathyrostachys huashanica amphiploids (PHW-SA, 2n = 8x = 56, AABBDDNsNs) with hexaploid triticale (Zhongsi 828, 2n = 6x = 42, AABBRR). In the present study, chromosome pairing behavior and the genome constitution of the F₄ progenies of wheat-rye-P. huashanica trigeneric hybrids were studied. Cytological analysis showed that the chromosome number of F₄ progenies ranged from 39 to 46, and 57.5% of them had 42 chromosomes. The mean meiotic configuration of F₄ lines was 1.71 univalents, 20.26 bivalents, 0.04 trivalents, and 0.001 quadrivalents per pollen mother cell. Among the lines with 2n = 42, the average pairing configuration was 1.21 univalents, 16.22 ring bivalents, 4.16 rod bivalents, and 0.01 trivalents. This result indicated that these lines were cytologically stable. Other lines with 2n = 39, 40, 41, 43, 44, 45, and 46, bearing a high number of univalents or multivalents, showed abnormal meiotic behavior. Genomic in situ hybridization (GISH) revealed that all F₄ lines had 11-14 rye chromosomes, but no P. huashanica chromosomes. The complete set of 14 rye chromosomes was found in 19 lines. At meiosis, GISH detected 1-6 univalents with hybridization signals of rye in 13 lines. Bivalents with fluorescence signals were identified in each line, ranging from 3 to 7. A quadrivalent with hybridization signals was observed in only 1 line, K13-714-8. Lagging chromosomes, chromosome bridges, micronuclei, and chromosome fragments hybridizing with the probe were not discovered in any of the lines. These results inferred that the behavior of rye chromosomes was normal during meiosis. In addition, 21 lines of 2n = 42 (91.3%) with 12 or 14 rye chromosomes, always contained 6 or 7 bivalents bearing fluorescence signals. This suggested that the rye chromosomes exhibiting complete pairing in these lines were cytologically stable during meiosis and may therefore be considered as new hexaploid triticales. Thus, these lines might be potential materials for further hexaploid triticale improvement.

CD30 Receptor-Targeted Lentiviral Vectors for Human Induced Pluripotent Stem Cell-Specific Gene Modification.

Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4(high) cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation, efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved, while retaining their pluripotency. When added during the reprogramming process, CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus, CD30-LV may serve as novel tool for the selective gene transfer into PSCs with broad applications in basic and therapeutic research.

Virulence Determinants among Extended-Spectrum B-Lactamases Producers of Uropathogenic Escherichia Coli Isolates in Zagagig University Hospitals, Egypt

Background: Uropathogenic E. coli (UPEC), the major causative agent of urinary tract infections (UTI) worldwide have variant virulent mechanisms. UPEC resistance to commonly used antibiotics represents a major health problem all over the world. The empiric antibacterial therapy of the infections should be based on a local experience of the susceptibility and the resistance profile. Our objectives are to detect the presence of and possible relation between virulence genes and ESBL production in E. coli strains isolated from patients with UTI in Zagazig, Egypt. Methodology: 75 E. coli strains isolated from patients with UTI were screened for virulence genes: fimH (type 1 fimbriae), pap (pyelonephritis-associated pili), sfa/foc (S and F1C fimbriae), afa (afimbrial adhesins), hly (hemolysin), cnf1 (cytotoxic necrotizing factor), aer (aerobactin), traT (transfer gene foe serum resistance) by polymerase chain reaction (PCR) and for antimicrobial resistance by disc diffusion method according to CLSI criteria along with extended spectrum B lactamases (ESBL) detection by double disc synergy (DDS) method. Results: The fimH gene was the most common virulence gene and was detected in 85.3% of the UTI isolates. The trat gene was present in 69.3% (52/75) and the pap gene in 52% of isolates. The aer gene was detected in 40% while the sfa gene and afa gene were shown in 29.3% and 21.3% of isolates respectively. Among the genes coding for toxin, hly was found in 9.3%, while cnf was present in only 8% of isolates. Eight strains genes lacked any tested virulence markers accounting (10.7%). Out of 75 strains, 41 (54.7%) were ESBL producers. Statistically significant relationships were found between fimH, tratT and pap with ESBL production (p values of <0.05, <0.0001 & <0.0001 respectively). Conclusions : There was a significant association between some virulence determinants of UPEC and ESBL production, these determinants may contribute to a better medical intervention. Also we need the used treatment regimens to be revised to reach better therapeutic effects .

Cytological aspects of incompatible and compatible interactions between rice, wheat and the blast pathogen Pyricularia oryzae

Blast, caused by the fungus Pyricularia oryzae, is an important disease affecting rice and wheat yield worldwide. This study investigated the cytological aspects of incompatible (non-host resistance) and compatible (host resistance) rice- (R_Po) and wheat- (W_Po) Pyricularia oryzae isolate interactions. Inoculations of rice and wheat with the R_Po and W_Po isolates of P. oryzae, respectively, were expected to be compatible interactions (host resistance), whereas inoculations of rice and wheat with the W_Po and R_Po isolates of P. oryzae, respectively, were considered to be incompatible interactions (non-host resistance). For the compatible interactions (rice-R_Po and wheat-W_Po), fungal hyphae penetrated and colonized the epidermal cells and also invaded many neighboring cells. By contrast, in the case of the incompatible interactions (rice-W_Po and wheat-R_Po), fungal hyphae were not able to penetrate nor colonize the epidermal cells, but when penetration did occur, the hyphae were restricted to the first-invaded epidermal cell. The frequency of appressorial sites exhibiting infection hyphae within the epidermal cell underlying an appressorium was greater in the case of the compatible interactions. By contrast, unsuccessful penetrations with cytoplasmic granulation occurred with high frequency in the incompatible wheat-R_Po and rice-W_Po interactions and the number of necrotic epidermal cells underlying the appressorium was low for the rice-W_Po interaction as well as for the wheat- R_Po interaction, where no symptoms of necrosis were exhibited. However, the opposite was observed for the compatible interactions. The present study presents cytological features associated with incompatible and compatible rice- and wheat-P. oryzae interactions that may be useful to studies involving variability, coevolution, diagnosis, and regulation of quarantine or even in a rice or wheat breeding program whose aim is to transfer genes involved in non-host resistance to host resistance due to similarities in downstream mechanisms.

The science (and art) of medicine

The science (and art) of medicine

Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain

Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer1-6. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics7-13. Here we describe a capsid selection method, called Cre-recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV914-17, and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications.

Impact of natural killer cells on chronic hepatitis C and hepatocellular carcinoma.

Natural killer (NK) cells are involved in the pathogenesis of hepatitis C viral (HCV) infection and hepatocellular carcinoma (HCC). Recent immunological progresses have revealed the molecular mechanisms of activation or inhibition of NK cells. In patients infected with HCV, the percentages of NK cells are decreased and the NK receptor expression and function of NK cells including cytotoxicity and cytokine production are altered. These alterations in NK cells are associated with persistent infection with HCV, liver injury, liver fibrosis and liver carcinogenesis. In HCV treatment, NK cells play a role in the eradication of HCV in both interferon (IFN)-based therapy and IFN-free therapy using direct-acting antivirals (DAA). In HCC patients, the exhaustion of NK cells that represents lower cytotoxicity and impaired cytokine production may contribute to the progression of HCC. Several immunotherapies targeting NK cells have been reported. NK cell transfer and NK-activating gene therapy have been demonstrated to be effective in mouse liver cancer models and several clinical trials are ongoing. Recently, the role of major histocompatibility complex class I-related chain A (MICA), a human ligand of NKG2D, has attracted attention in the development of HCC. The expression of MICA could be controlled by anti-HCC drugs including sorafenib. A new chemo-immunotherapy may be expected in the treatment of HCC. In this review, we summarize the impact of NK cells on chronic hepatitis C and HCC.

Two different routes for double-stranded DNA transfer in natural and artificial transformation of Escherichia coli

Escherichia coli is naturally transformable, independent on the conserved DNA uptake machinery for single-stranded DNA (ssDNA) integration. The transfer of double-stranded DNA (dsDNA) during natural transformation of E. coli is regulated by the alternative sigma factor σS. However, it remains mysterious how dsDNA transfers across the membranes and how σS regulates natural transformation of E. coli. Here, I screened for σS-regulated genes for dsDNA transfer in E. coli. The screening identified the σS-regulated genes ydcS and ydcV, both locate on the putative ABC transporter ydcSTUV operon. Considering that ydcS and ydcV are predicted to encode a periplasmic protein and an inner membrane protein for substrate binding and translocation respectively, I propose that they may mediate dsDNA translocation across the inner membrane during natural transformation. In chemical transformation of E. coli, ydcS was but ydcV was not required. Thus, YdcV should not be the channel for dsDNA translocation in artificial transformation. Together with the previous observation that the outer membrane porin OmpA mediates dsDNA transfer across the outer membrane in chemical transformation but not in natural transformation, I conclude that dsDNA transfers across the two membranes through different routes in natural and artificial transformation of E. coli.