Karyotype engineering by chromosome fusion leads to reproductive isolation in yeast.

Extant species have wildly different numbers of chromosomes, even among taxa with relatively similar genome sizes (for example, insects)1,2. This is likely to reflect accidents of genome history, such as telomere-telomere fusions and genome duplication events3-5. Humans have 23 pairs of chromosomes, whereas other apes have 24. One human chromosome is a fusion product of the ancestral state6. This raises the question: how well can species tolerate a change in chromosome numbers without substantial changes to genome content? Many tools are used in chromosome engineering in Saccharomyces cerevisiae7-10, but CRISPR-Cas9-mediated genome editing facilitates the most aggressive engineering strategies. Here we successfully fused yeast chromosomes using CRISPR-Cas9, generating a near-isogenic series of strains with progressively fewer chromosomes ranging from sixteen to two. A strain carrying only two chromosomes of about six megabases each exhibited modest transcriptomic changes and grew without major defects. When we crossed a sixteen-chromosome strain with strains with fewer chromosomes, we noted two trends. As the number of chromosomes dropped below sixteen, spore viability decreased markedly, reaching less than 10% for twelve chromosomes. As the number of chromosomes decreased further, yeast sporulation was arrested: a cross between a sixteen-chromosome strain and an eight-chromosome strain showed greatly reduced full tetrad formation and less than 1% sporulation, from which no viable spores could be recovered. However, homotypic crosses between pairs of strains with eight, four or two chromosomes produced excellent sporulation and spore viability. These results indicate that eight chromosome-chromosome fusion events suffice to isolate strains reproductively. Overall, budding yeast tolerates a reduction in chromosome number unexpectedly well, providing a striking example of the robustness of genomes to change.

Creating a functional single-chromosome yeast.

Eukaryotic genomes are generally organized in multiple chromosomes. Here we have created a functional single-chromosome yeast from a Saccharomyces cerevisiae haploid cell containing sixteen linear chromosomes, by successive end-to-end chromosome fusions and centromere deletions. The fusion of sixteen native linear chromosomes into a single chromosome results in marked changes to the global three-dimensional structure of the chromosome due to the loss of all centromere-associated inter-chromosomal interactions, most telomere-associated inter-chromosomal interactions and 67.4% of intra-chromosomal interactions. However, the single-chromosome and wild-type yeast cells have nearly identical transcriptome and similar phenome profiles. The giant single chromosome can support cell life, although this strain shows reduced growth across environments, competitiveness, gamete production and viability. This synthetic biology study demonstrates an approach to exploration of eukaryote evolution with respect to chromosome structure and function.

Central Nervous System Delivery and Biodistribution Analysis of an Antibody-Enzyme Fusion for the Treatment of Lafora Disease.

Lafora disease (LD) is a fatal juvenile epilepsy characterized by the accumulation of aberrant glucan aggregates called Lafora bodies (LBs). Delivery of protein-based therapeutics to the central nervous system (CNS) for the clearance of LBs remains a unique challenge in the field. Recently, a humanized antigen-binding fragment (hFab) derived from a murine systemic lupus erythematosus DNA autoantibody (3E10) has been shown to mediate cell penetration and proposed as a broadly applicable carrier to mediate cellular targeting and uptake. We report studies on the efficacy and CNS delivery of VAL-0417, an antibody-enzyme fusion composed of the 3E10 hFab and human pancreatic α-amylase, in a mouse model of LD. An enzyme-linked immunosorbent assay has been developed to detect VAL-0417 post-treatment as a measure of delivery efficacy. We demonstrate the robust and sensitive detection of the fusion protein in multiple tissue types. Using this method, we measured biodistribution in different methods of delivery. We found that intracerebroventricular administration provided robust CNS delivery when compared to intrathecal administration. These data define critical steps in the translational pipeline of VAL-0417 for the treatment of LD.

Generation of conditional oncogenic chromosomal translocations using CRISPR-Cas9 genomic editing and homology-directed repair.

Chromosomal rearrangements encoding oncogenic fusion proteins are found in a wide variety of malignancies. The use of programmable nucleases to generate specific double-strand breaks in endogenous loci, followed by non-homologous end joining DNA repair, has allowed several of these translocations to be generated as constitutively expressed fusion genes within a cell population. Here, we describe a novel approach that combines CRISPR-Cas9 technology with homology-directed repair to engineer, capture, and modulate the expression of chromosomal translocation products in a human cell line. We have applied this approach to the genetic modelling of t(11;22)(q24;q12) and t(11;22)(p13;q12), translocation products of the EWSR1 gene and its 3' fusion partners FLI1 and WT1, present in Ewing's sarcoma and desmoplastic small round cell tumour, respectively. Our innovative approach allows for temporal control of the expression of engineered endogenous chromosomal rearrangements, and provides a means to generate models to study tumours driven by fusion genes. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Construction and expression of prokaryotic expression vectors fused with genes of Magnaporthe oryzae effector proteins and mCherry.

The aim of the current study was to investigate the prokaryotic expression of the Magnaporthe oryzae effector genes BAS1 and BAS4 fused to the fluorescent protein mCherry. Based on previous polymorphic analysis of BAS1 and BAS4 in rice blast strains using PCR, blast strains containing the PCR products of BAS1 and BAS4 were selected for liquid culture for total RNA extraction. For PCR analysis, cDNA was selected as a template to amplify the coding region of BAS1 and BAS4, the plasmid pXY201 was selected as template to amplify the mCherry sequence, and the three sequences were cloned into pMD®19-T vectors. Positive recombinant plasmids were digested using two restriction enzymes and the cleaved fragments of BAS1 and mCherry and BAS4 and mCherry were ligated to pGEX-4T-1 vectors and expression was induced using IPTG. The PCR results showed that the sequence sizes of BAS1, BAS4, and mCherry were 348, 309, and 711 bp, respectively, and these were cloned into pMD®19-T vectors. After digestion and gel purification, the fragments of BAS1 and mCherry, BAS4 and mCherry were ligated into pGEX-4T-1 vectors and expressed in Escherichia coli BL21 competent cells. The expressed proteins were approximately 60 kDa, corresponding to their theoretical size. Prokaryotic expression products of BAS1 and BAS4 fused to mCherry were presented in this study, providing a base for constructing prokaryotic expression vectors of pathogen effector genes fused to mCherry, which will contribute to further study of the subcellular localization, function, and protein interactions of these effectors.

Chimeric murine interferon regulatory factor-2 (IRF-2) binds to IRF-E (IRF binding element), VREß (virus response element) but not to VREα1.

Interferon regulatory factor-2 (IRF-2) is a multifunctional transcription factor having gene activation, repression and synergistic effect in conjunction with IRF-1. IRF-2 is also involved in type I IFN signalling by repressing INFß gene. So far, the molecular mechanism of its DNA binding activity remains elusive. We have carried out molecular sub-cloning, expression and electrophoretically mobility shift assay study of chimeric murine IRF-2. Here, we report expression of chimeric murine IRF-2 as GST-IRF-2 fusion protein in Escherichia coli/BL21 cells and demonstrated DNA binding activity by gel retardation technique using radio (32) P-labelled IRF-E motif (GAAAGT)4 , virus response element (VRE) of human INFß and IFNα1 gene. We observed five different masses DNA/GST-IRF-2 complexes (1-5) with IRF-E motif, three different masses DNA/GST-IRF-2 complexes (1-3) with VREß , but we could not observe any complex of DNA/GST-IRF-2 with VREα1 . The specific binding on IRF-E motif was confirmed by carrying out 100-X fold cold competition with (32) P-labelled IRF-E motif. In contrast to specific binding on VREß , we used negative control where we observed no binding complex, but we observed complexes with clones IPTG-induced extract. As far as binding on VREα1 is concerned, we could not observe any complex in negative control as well as in IPTG-inducible clones extract. Chimeric IRF-2 binds with IRF-E motif and VREß but not with VREα1. This study is first of its kind and paves the way to understand the differential DNA binding and molecular mechanism of DNA binding activity of the IRF-2 molecule, which is crucial for its function(s).

Design and optimization of short DNA sequences that can be used as 5' fusion partners for high-level expression of heterologous genes in Escherichia coli.

The 5' terminal nucleotide sequence of a gene is often a bottleneck in recombinant protein production. The ifn-α2bS gene is poorly expressed in Escherichia coli unless a translocation signal sequence (pelB) is fused to the 5' end of the gene. A combined in silico and in vivo analysis reported here further indicates that the ifn-α2bS 5' coding sequence is suboptimal for efficient gene expression. ifn-α2bS therefore presents a suitable model gene for describing properties of 5' fusions promoting expression. We show that short DNA sequences corresponding to the 5' end of the highly expressed celB gene, whose protein product is cytosolic, can functionally replace pelB as a 5' fusion partner for efficient ifn-α2bS expression. celB fusions of various lengths (corresponding to a minimum of 8 codons) led to more than 7- and 60-fold stimulation of expression at the transcript and protein levels, respectively. Moreover, the presence of a celB-based fusion partner was found to moderately reduce the decay rate of the corresponding transcript. The 5' fusions thus appear to act by enhancing translation, and bound ribosomes may accordingly contribute to increased mRNA stability and reduced mRNA decay. However, other effects, such as altered protein stability, cannot be excluded. We also developed an experimental protocol that enabled us to identify improved variants of the celB fusion, and one of these (celBD11) could be used to additionally increase ifn-α2bS expression more than 4-fold at the protein level. Interestingly, celBD11 also stimulated greater protein production of three other medically important human genes than the wild-type celB fragment.

The Y chromosome of the Okinawa spiny rat, Tokudaia muenninki, was rescued through fusion with an autosome.

The genus Tokudaia comprises three species, two of which have lost their Y chromosome and have an XO/XO sex chromosome constitution. Although Tokudaia muenninki (Okinawa spiny rat) retains the Y chromosome, both sex chromosomes are unusually large. We conducted a molecular cytogenetic analysis to characterize the sex chromosomes of T. muenninki. Using cross-species fluorescence in situ hybridization (Zoo-FISH), we found that both short arms of the T. muenninki sex chromosomes were painted by probes from mouse chromosomes 11 and 16. Comparative genomic hybridization analysis was unable to detect sex-specific regions in the sex chromosomes because both sex probes highlighted the large heterochromatic blocks on the Y chromosome as well as five autosomal pairs. We then performed comparative FISH mapping using 29 mouse complementary DNA (cDNA) clones of the 22 X-linked genes and the seven genes linked to mouse chromosome 11 (whose homologue had fused to the sex chromosomes), and FISH mapping using two T. muenninki cDNA clones of the Y-linked genes. This analysis revealed that the ancestral gene order on the long arm of the X chromosome and the centromeric region of the short arm of the Y chromosome were conserved. Whereas six of the mouse chromosome 11 genes were also mapped to Xp and Yp, in addition, one gene, CBX2, was also mapped to Xp, Yp, and chromosome 14 in T. muenninki. CBX2 is the candidate gene for the novel sex determination system in the two other species of Tokudaia, which lack a Y chromosome and SRY gene. Overall, these results indicated that the Y chromosome of T. muenninki avoided a loss event, which occurred in an ancestral lineage of T. osimensis and T. tokunoshimensis, through fusion with an autosome. Despite retaining the Y chromosome, sex determination in T. muenninki might not follow the usual mammalian pattern and deserves further investigation.

End-to-end gene fusions and their impact on the production of multifunctional biomass degrading enzymes.

The reduction of fossil fuels, coupled with its increase in price, has made the search for alternative energy resources more plausible. One of the topics gaining fast interest is the utilization of lignocellulose, the main component of plants. Its primary constituents, cellulose and hemicellulose, can be degraded by a series of enzymes present in microorganisms, into simple sugars, later used for bioethanol production. Thermophilic bacteria have proven to be an interesting source of enzymes required for hydrolysis since they can withstand high and denaturing temperatures, which are usually required for processes involving biomass degradation. However, the cost associated with the whole enzymatic process is staggering. A solution for cost effective and highly active production is through the construction of multifunctional enzyme complexes harboring the function of more than one enzyme needed for the hydrolysis process. There are various strategies for the degradation of complex biomass ranging from the regulation of the enzymes involved, to cellulosomes, and proteins harboring more than one enzymatic activity. In this review, the construction of multifunctional biomass degrading enzymes through end-to-end gene fusions, and its impact on production and activity by choosing the enzymes and linkers is assessed.

A gene-fusion approach to enabling plant cytochromes p450 for biocatalysis.

Cytochromes P450 from plants have the potential to be valuable catalysts for industrial hydroxylation reactions, but their application is hindered by poor solubility, the lack of suitable expression systems and the requirement of P450s for auxiliary redox-transport proteins for the delivery of reducing equivalents from NAD(P)H. In the interests of enabling useful P450 activity from plants, we have developed a suite of vectors for the expression of plant P450s as non-natural genetic fusions with reductase proteins. First, we have fused the P450 isoflavone synthase (IFS) from Glycine max with the bacterial P450 reductase domain (Rhf-RED) from Rhodococcus sp., by using our LICRED vector developed previously (F. Sabbadin, R. Hyde, A. Robin, E.-M. Hilgarth, M. Delenne, S. Flitsch, N. Turner, G. Grogan, N. C. Bruce, ChemBioChem 2010, 11, 987-994) creating the first active bacterial-plant fusion P450 enzyme. We have then created a complementary vector, ACRyLIC for the fusion of selected plant P450 enzymes to the P450 reductase ATR2 from Arabidopsis thaliana. The applicability of this vector to the creation of active P450 fusion enzymes was demonstrated using both IFS1 and the cinnamate-4-hydroxylase (C4H) from A. thaliana. Overall the fusion vector systems will allow the rapid creation of libraries of plant P450s with the aim of identifying enzyme activities with possible applications in industrial biocatalysis.

One-pot fusion polymerase chain reaction for combinatorial synthesis of DNA from several cassettes.

The fusion of DNA fragments is becoming increasingly more important. The ability to work without being constrained by restriction sites enables DNA fusion to be applied to a much broader range of fragments. Therefore, we describe a simplified polymerase chain reaction (PCR)-based method for fusion of DNA fragments in one step. In a single PCR, two templates, an overlapping primer, and template-specific forward and reverse primers are used. After a few cycles, the fusion DNA is assembled and is amplified. The ratio of overlapping primer to forward/reverse primers and template DNA is essential for the success of the reaction.

[Cell surface display of Thermomyces lanuginosus lipase in Pichia pastoris and its characterization].

OBJECTIVE: To construct a novel cell-surface display system of Thermomyces lanuginosus lipase (TLL) based on an efficient anchor protein Sedlp in Pichia pastoris, to screen recombinant strains with high enzyme activity and displaying rate, and further to characterize the enzyme. METHODS: The lipase gene from T. lanuginosus was sub-cloned and fused with the anchor protein gene sed1 from Saccharomyces cerevisiae to construct a display vector pPICZalphaA-TLS. The vector pPICZalphaA-TLS was linearized by Sac I and then transformed into P. pastoris GS115 by electroporation. After screening by tributyrin medium, a clone exhibiting the maximum lipase activity in shaking flask was chosen to treat with rabbit anti-FLAG-tag and R-PE-conjugated goat anti-rabbit IgG, and then its positive location on the cell wall was detected by fluorescence microscope and flow cytometer. The recombinant strain displaying TLL was further characterized as a whole-cell catalyst. RESULTS: A novel cell-surface display system of T. lanuginosus lipase was successfully established, and a clone with lipase activity of 257.8 U/g dry cells in shaking flask was obtained. The displayed TLL on the cell surface was confirmed by immunofluorescence, and the treated cells under the fluorescence microscope emitted brightly red fluorescence, and the displaying rate was 98.36% detected by Flow Cytometer. The displayed TLL exhibited excellent thermostability and high tolerance to some organic solvents, and its maximal activity was observed at 30 degrees C and pH 8.0. The lipase activity was a little enhanced by K+, Ca2+ and Mg2+ and strongly inhibited by Cu2+, Mn2+ and Ni2+. However, ethylenediaminetetraacetic acid (EDTA), Sodium lauryl sulfate (SDS) and Tween 20 showed little effect on the displayed TLL. CONCLUSION: The lipase TLL was successfully displayed on the cell surface of P. pastoris by the anchor protein Sed1p for the first time to obtain a whole-cell catalyst, which had high hydrolytic activity and excellent enzymatic characterization. Thus, we here established a solid foundation for industrial applications of the displayed lipase TLL.

A sustainable mouse karyotype created by programmed chromosome fusion.

Chromosome engineering has been attempted successfully in yeast but remains challenging in higher eukaryotes, including mammals. Here, we report programmed chromosome ligation in mice that resulted in the creation of new karyotypes in the lab. Using haploid embryonic stem cells and gene editing, we fused the two largest mouse chromosomes, chromosomes 1 and 2, and two medium-size chromosomes, chromosomes 4 and 5. Chromatin conformation and stem cell differentiation were minimally affected. However, karyotypes carrying fused chromosomes 1 and 2 resulted in arrested mitosis, polyploidization, and embryonic lethality, whereas a smaller fused chromosome composed of chromosomes 4 and 5 was able to be passed on to homozygous offspring. Our results suggest the feasibility of chromosome-level engineering in mammals.

Directed chromosomal integration and expression of the reporter gene gusA3 in Lactobacillus acidophilus NCFM.

Lactobacillus acidophilus NCFM is a probiotic microbe that survives passage through the human gastrointestinal tract and interacts with the host epithelium and mucosal immune cells. The potential for L. acidophilus to express antigens at mucosal surfaces has been investigated with various antigens and plasmid expression vectors. Plasmid instability and antibiotic selection complicate the possibility of testing these constructs in human clinical trials. Integrating antigen encoding genes into the chromosome for expression is expected to eliminate selection requirements and provide genetic stability. In this work, a reporter gene encoding a ß-glucuronidase (GusA3) was integrated into four intergenic chromosomal locations. The integrants were tested for genetic stability and GusA3 activity. Two locations were selected for insertion downstream of constitutively highly expressed genes, one downstream of slpA (LBA0169), encoding a highly expressed surface-layer protein, and one downstream of phosphopyruvate hydratase (LBA0889), a highly expressed gene with homologs in other lactic acid bacteria. An inducible location was selected downstream of lacZ (LBA1462), encoding a ß-galactosidase. A fourth location was selected in a low-expression region. The expression of gusA3 was evaluated from each location by measuring GusA3 activity on 4-methyl-umbelliferyl-ß-d-glucuronide (MUG). GusA3 activity from both highly expressed loci was more than three logs higher than the gusA3-negative parent, L. acidophilus NCK1909. GusA3 activity from the lacZ locus was one log higher in cells grown in lactose than in glucose. The differences in expression levels between integration locations highlights the importance of rational targeting with gene cassettes intended for chromosomal expression.

Alternative Seamless Cloning Strategies in Fusing Gene Fragments Based on Overlap-PCR.

Gene fragment swapping and site-directed mutagenesis are commonly required in dissecting functions of gene domains. While there are many approaches for seamless fusion of different gene fragments, new methods are yet to be developed to offer higher efficiency, better simplicity, and more affordability. In this study, we showed that in most cases overlap-PCR was highly effective in creating site-directed mutagenesis, gene fragment deletion, and substitutions using RUS1 and RUS2 as example. While for cases where the overlap-PCR approach is not feasible due to complex secondary structure of gene fragments, a unique restriction site can be generated at the overlapped region of the primers through synonymous mutations. Then different gene fragments can be seamlessly fused through traditional restriction digestion and subsequent ligation. In conclusion, while the classical overlap-PCR is not feasible, the modified overlap-PCR approaches can provide effective and alternative ways to seamlessly fuse different gene fragments.

Molecular dissection of ALS-associated toxicity of SOD1 in transgenic mice using an exon-fusion approach.

Mutations in Cu,Zn superoxide dismutase (SOD1) are associated with amyotrophic lateral sclerosis (ALS). Among more than 100 ALS-associated SOD1 mutations, premature termination codon (PTC) mutations exclusively occur in exon 5, the last exon of SOD1. The molecular basis of ALS-associated toxicity of the mutant SOD1 is not fully understood. Here, we show that nonsense-mediated mRNA decay (NMD) underlies clearance of mutant mRNA with a PTC in the non-terminal exons. To further define the crucial ALS-associated SOD1 fragments, we designed and tested an exon-fusion approach using an artificial transgene SOD1(T116X) that harbors a PTC in exon 4. We found that the SOD1(T116X) transgene with a fused exon could escape NMD in cellular models. We generated a transgenic mouse model that overexpresses SOD1(T116X). This mouse model developed ALS-like phenotype and pathology. Thus, our data have demonstrated that a 'mini-SOD1' of only 115 amino acids is sufficient to cause ALS. This is the smallest ALS-causing SOD1 molecule currently defined. This proof of principle result suggests that the exon-fusion approach may have potential not only to further define a shorter ALS-associated SOD1 fragment, thus providing a molecular target for designing rational therapy, but also to dissect toxicities of other proteins encoded by genes of multiple exons through a 'gain of function' mechanism.

A novel method for promoting heterologous protein expression in Escherichia coli by fusion with the HIV-1 TAT core domain.

The human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (TAT) protein, a member of the protein transduction domain (PTD) superfamily, can deliver heterologous proteins across most biomembranes without losing bioactivity. However, there is no report on whether the TAT core domain containing the sequence 'YGRKKRRQRRR' has other functions. As the TAT core domain is most basic (pI=12.8) and has biomembrane crossing ability, we hypothesized it might probably influence the protein expression level due to subcellular redistribution of target proteins in the cells. To address this issue, we constructed the prokaryotic expression vector pET28b-TAT-EGFP (using the vector pET28b-EGFP for control) containing the core domain coding region, and transformed the vector into E. coli BL21 (DE3) cells for expression of the enhanced green fluorescent protein (EGFP) with the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). Equal amount of the total proteins were fractionated using 15% SDS-PAGE and identified by western blot, and the plasmid copy number was assayed by Southern blot. In order to further study the subcellular localization of heterologous proteins in E. coli cells, the cytoplasmic and periplasmic components were extracted by chloroform and osmotic shock techniques. Interestingly, our data showed that the TAT core domain was not only able to promote the heterologous protein expression in E. coli, but also improve the yields and the solubility of heterologous proteins, while the plasmid copy number of TAT-containing clones and TAT-free clones was not affected by the TAT core domain. In addition, the TAT-tagged protein was mainly localized in the cytoplasm and also accumulated in the periplasmic space along with the time for protein expression, while in contrast, the TAT-free protein was mainly expressed in the periplasm and only a few in cytoplasm. A further examination on the distribution of the expressed proteins in cytoplasm and periplasm suggested that the TAT core domain might promote protein expression in the cytoplasm initially and then partially deliver them across the cytomembrane to the periplasmic space in a concentration-dependent manner. Taken together, our current data have provided a novel method for improving heterologous protein expression in prokaryotic cells by fusion with the TAT core domain, which will promote expression efficiency of bioactive proteins for protein engineering.

Simple and effective gap-repair cloning using short tracts of flanking homology in fission yeast.

Gap-repair cloning for plasmid construction in budding yeast is very effective and often used. In contrast, the same method is not widely used in fission yeast, because of a shortage of information on it. Here we describe simple and effective gap-repair cloning for plasmid construction using short tracts of flanking homology. By this method, we combined concentrated DNA fragments with short (20 bp) tracts of flanking homology with the marker gene or the pre-existing gene module. In addition, we found that this method can be applied to one-step cloning of multiple DNA fragments to construct a fusion gene.

XCL1/Glypican-3 Fusion Gene Immunization Generates Potent Antitumor Cellular Immunity and Enhances Anti-PD-1 Efficacy.

Cancer vaccines can amplify existing antitumor responses or prime naïve T cells to elicit effector T-cell functions in patients through immunization. Antigen-specific CD8+ T cells are crucial for the rejection of established tumors. We constructed XCL1-GPC3 fusion molecules as a liver cancer vaccine by linking the XCL1 chemokine to glypican-3 (GPC3), which is overexpressed in hepatocellular carcinoma (HCC). Cells expressing XCL1-GPC3 chemoattracted murine XCR1+CD8α+ dendritic cells (DC) and human XCR1+CD141+ DCs in vitro and promoted their IL12 production. After subcutaneous mXcl1-GPC3 plasmid injection, mXCL1-GPC3 was mainly detected in CD8α+ DCs of mouse draining lymph nodes. XCL1-GPC3-targeted DCs enhanced antigen-specific CD8+ T-cell proliferation and induced the de novo generation of GPC3-specific CD8+ T cells, which abolished GPC3-expressing tumor cells in mouse and human systems. We immunized a murine autochthonous liver cancer model, with a hepatitis B background, with the mXcl1-GPC3 plasmid starting at 6 weeks, when malignant hepatocyte clusters formed, or at 14 weeks, when liver tumor nodules developed, after diethylnitrosamine administration. mXcl1-GPC3-immunized mice displayed significantly inhibited tumor formation and growth compared with GPC3-immunized mice. After mXcl1-GPC3 immunization, mouse livers showed elevated production of IFNγ, granzyme B, IL18, CCL5, CXCL19, and Xcl1 and increased infiltration of GPC3-specific CD8+ T cells, activated natural killer (NK) cells, and NKT cells. The antitumor effects of these immune cells were further enhanced by the administration of anti-PD-1. Anti-HCC effects induced by hXCL1-GPC3 were confirmed in an HCC-PDX model from 3 patients. Thus, XCL1-GPC3 might be a promising cancer vaccine to compensate for the deficiency of the checkpoint blockades in HCC immunotherapy.

A multifunctional, synthetic Gaussia princeps luciferase reporter for live imaging of Candida albicans infections.

Real-time monitoring of the spatial and temporal progression of infection/gene expression in animals will contribute greatly to our understanding of host-pathogen interactions while reducing the number of animals required to generate statistically significant data sets. Sensitive in vivo imaging technologies can detect low levels of light emitted from luciferase reporters in vivo, but the existing reporters are not optimal for fungal infections. Therefore, our aim was to develop a novel reporter system for imaging Candida albicans infections that overcomes the limitations of current luciferase reporters for this major fungal pathogen. This luciferase reporter was constructed by fusing a synthetic, codon-optimized version of the Gaussia princeps luciferase gene to C. albicans PGA59, which encodes a glycosylphosphatidylinositol-linked cell wall protein. Luciferase expressed from this PGA59-gLUC fusion (referred to as gLUC59) was localized at the C. albicans cell surface, allowing the detection of luciferase in intact cells. The analysis of fusions to strong (ACT1 and EFT3), oxidative stress-induced (TRX1, TRR1, and IPF9996), and morphogenesis-dependent (HWP1) promoters confirmed that gLUC59 is a convenient and sensitive reporter for studies of gene regulation in yeast or hyphal cells, as well as a flexible screening tool. Moreover, the ACT1-gLUC59 fusion represented a powerful tool for the imaging of disease progression in superficial and subcutaneous C. albicans infections. gLUC59 and related cell surface-exposed luciferase reporters might find wide applications in molecular biology, cell biology, pathobiology, and high-throughput screens.