Uracil Phosphoribosyltransferase Research Articles

Enhanced anti-tumor efficacy with multi-transgene armed mesenchymal stem cells for treating peritoneal carcinomatosis

BackgroundMesenchymal stem cells (MSCs) have garnered significant interest for their tumor-tropic property, making them potential therapeutic delivery vehicles for cancer treatment. We have previously shown the significant anti-tumour activity in mice preclinical models and companion animals with naturally occurring cancers using non-virally engineered MSCs with a therapeutic transgene encoding cytosine deaminase and uracil phosphoribosyl transferase (CDUPRT) and green fluorescent protein (GFP). Clinical studies have shown improved response rate with combinatorial treatment of 5-fluorouracil and Interferon-beta (IFNb) in peritoneal carcinomatosis (PC). However, high systemic toxicities have limited the clinical use of such a regime.MethodsIn this study, we evaluated the feasibility of intraperitoneal administration of non-virally engineered MSCs to co-deliver CDUPRT/5-Flucytosine prodrug system and IFNb to potentially enhance the cGAS-STING signalling axis. Here, MSCs were engineered to express CDUPRT or CDUPRT-IFNb. Expression of CDUPRT and IFNb was confirmed by flow cytometry and ELISA, respectively. The anti-cancer efficacy of the engineered MSCs was evaluated in both in vitro and in vivo model. ES2, HT-29 and Colo-205 were cocultured with engineered MSCs at various ratio. The cell viability with or without 5-flucytosine was measured with MTS assay. To further compare the anti-cancer efficacy of the engineered MSCs, peritoneal carcinomatosis mouse model was established by intraperitoneal injection of luciferase expressing ES2 stable cells. The tumour burden was measured through bioluminescence tracking.ResultsFirstly, there was no changes in phenotypes of MSCs despite high expression of the transgene encoding CDUPRT and IFNb (CDUPRT-IFNb). Transwell migration assays and in-vivo tracking suggested the co-expression of multiple transgenes did not impact migratory capability of the MSCs. The superiority of CDUPRT-IFNb over CDUPRT expressing MSCs was demonstrated in ES2, HT-29 and Colo-205 in-vitro. Similar observations were observed in an intraperitoneal ES2 ovarian cancer xenograft model. The growth of tumor mass was inhibited by ~ 90% and 46% in the mice treated with MSCs expressing CDUPRT-IFNb or CDUPRT, respectively.ConclusionsTaken together, these results established the effectiveness of MSCs co-expressing CDUPRT and IFNb in controlling and targeting PC growth. This study lay the foundation for the development of clinical trial using multigene-armed MSCs for PC.

SLAM-ITseq identifies that Nrf2 induces liver regeneration through the pentose phosphate pathway

The liver exhibits a remarkable capacity to regenerate following injury. Despite this unique attribute, toxic injury is a leading cause of liver failure. The temporal processes by which the liver senses injury and initiates regeneration remain unclear. Here, we developed a transgenic zebrafish model wherein hepatocyte-specific expression of uracil phosphoribosyltransferase (UPRT) enabled the implementation of SLAM-ITseq to investigate the nascent transcriptome during initiation of liver injury and regeneration. Using this approach, we identified a rapid metabolic transition from the fed to the fasted state that was followed by induction of the nuclear erythroid 2-related factor (Nrf2) antioxidant program. We find that activation of Nrf2 in hepatocytes is required to induce the pentose phosphate pathway (PPP) and improve survival following liver injury. Mechanistically, we demonstrate that inhibition of the PPP disrupts nucleotide biosynthesis to prevent liver regeneration. Together, these studies provide fundamental insights into the mechanism by which early metabolic adaptation to injury facilitates tissue regeneration.

Harnessing the endogenous Type I-C CRISPR-Cas system for genome editing in Bifidobacterium breve.

Bifidobacterium breve, one of the main bifidobacterial species colonizing the human gastrointestinal tract in early life, has received extensive attention for its purported beneficial effects on human health. However, exploration of the mode of action of such beneficial effects exerted by B. breve is cumbersome due to the lack of effective genetic tools, which limits its synthetic biology application. The widespread presence of CRISPR-Cas systems in the B. breve genome makes endogenous CRISPR-based gene editing toolkits a promising tool. This study revealed that Type I-C CRISPR-Cas systems in B. breve can be divided into two groups based on the amino acid sequences encoded by cas gene clusters. Deletion of the gene coding uracil phosphoribosyl-transferase (upp) was achieved in five B. breve strains from both groups using this system. In addition, translational termination of uracil phosphoribosyl-transferase was successfully achieved in B. breve FJSWX38M7 by single-base substitution of the upp gene and insertion of three stop codons. The gene encoding linoleic acid isomerase (bbi) in B. breve, being a characteristic trait, was deleted after plasmid curing, which rendered it unable to convert linoleic acid into conjugated linoleic acid, demonstrating the feasibility of successive editing. This study expands the toolkit for gene manipulation in B. breve and provides a new approach toward functional genome editing and analysis of B. breve strains.IMPORTANCEThe lack of effective genetic tools for Bifidobacterium breve is an obstacle to studying the molecular mechanisms of its health-promoting effects, hindering the development of next-generation probiotics. Here, we introduce a gene editing method based on the endogenous CRISPR-Cas system, which can achieve gene deletion, single-base substitution, gene insertion, and successive gene editing in B. breve. This study will facilitate discovery of functional genes and elucidation of molecular mechanisms of B. breve pertaining to health-associated benefits.

Suicide-Gene-Modified Extracellular Vesicles of Human Primary Uveal Melanoma in Future Therapies.

Extracellular vesicles secreted from uveal melanoma (UM) cells are involved in the establishment of the premetastatic niche and display transforming potential for the formation of metastases, preferentially in the liver. In this study, we cultivated human primary UM cells and uveal melanoma-associated fibroblasts in vitro to be transduced by infection with a retrovirus containing the suicide gene-fused yeast cytosine deaminase::uracil phospho-ribosyl transferase (yCD::UPRT). A homogenous population of yCD::UPRT-UM cells with the integrated provirus expressed the gene, and we found it to continuously secrete small extracellular vesicles (sEVs) possessing mRNA of the suicide gene. The yCD::UPRT-UM-sEVs were internalized by tumor cells to the intracellular conversion of the prodrug 5-fluorocytosine (5-FC) to the cytotoxic drug 5-fluorouracil (5-FU). The host range of the yCD::UPRT-UM-sEVs was not limited to UMs only. The yCD::UPRT-UM-sEVs inhibited the growth of the human cutaneous melanoma cell line A375 and uveal melanoma cell line MP38, as well as other primary UMs, to various extents in vitro. The yCD::UPRT-UM-sEVs hold the therapeutic and prophylactic potential to become a therapeutic drug for UM. However, the use of yCD::UPRT-UM-sEVs must first be tested in animal preclinical studies.

A qPCR Method to Assay Endonuclease Activity of Cas9-sgRNA Ribonucleoprotein Complexes.

The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/μg RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.

Development of an efficient ClosTron system for gene disruption in Ruminiclostridium papyrosolvens.

Ruminiclostridium papyrosolvens is an anaerobic, mesophilic, and cellulolytic clostridia, promising consolidated bioprocessing (CBP) candidate for producing renewable green chemicals from cellulose, but its metabolic engineering is limited by lack of genetic tools. Here, we firstly employed the endogenous xylan-inducible promoter to control ClosTron system for gene disruption of R. papyrosolvens. The modified ClosTron can be easily transformed into R. papyrosolvens and specifically disrupt targeting genes. Furthermore, a counter selectable system based on uracil phosphoribosyl-transferase (Upp) was successfully established and introduced into the ClosTron system, which resulted in plasmid curing rapidly. Thus, the combination of xylan-inducible ClosTron and upp-based counter selectable system makes the gene disruption more efficient and convenient for successive gene disruption in R. papyrosolvens. KEY POINTS: • Limiting expression of LtrA enhanced the transformation of ClosTron plasmids in R. papyrosolvens. • DNA targeting specificity can be improved by precise management of the expression of LtrA. • Curing of ClosTron plasmids was achieved by introducing the upp-based counter selectable system.

Evaluation of nanoplastics toxicity to the human placenta in systems

Following the discovery of plastics in the human placenta, this study evaluated the toxicity of ten different nanoplastics (NPs) in the human placenta. Since the placenta performs metabolic and excretion functions by the enzymatic system, the NPs were docked on these human enzymes including soluble epoxide hydrolase, uracil phosphoribosyltransferase, beta 1,3-glucuronyltransferase I, sulfotransferase, N-acetyltransferase 2, and cytochrome P450 1A1at their active sites with toxicity (binding affinity) determined and compared to control compounds. Density functional theory analysis were conducted on the NPs to identify their global reactivity descriptors and Artificial Neural Networks to predict toxicity based on reactivity descriptors. Polycarbonate (PC), polyethylene terephthalate (PET) and polystyrene (PS) showed the highest toxicity to all enzymes and thus the most toxic polymers due to the presence of an electron-withdrawing group in their aromatic rings, which demonstrated an improved recognition of the enzyme active site by pi- and alkyl interactions. A 210–6 fractional factorial design approach was used in conjunction with a fixed effects model to assess the primary and secondary effects of NPs in a composite system on binding affinity to the placental enzymes. The simulation results suggest that NPs mixture may pose significant risks to the placenta through inhibition of its key enzymes.

An Optimized Enzyme-Nucleobase Pair Enables In Vivo RNA Metabolic Labeling with Improved Cell-Specificity.

Current transcriptome-wide analyses have identified a growing number of regulatory RNA with expression that is characterized in a cell-type-specific manner. Herein, we describe RNA metabolic labeling with improved cell-specificity utilizing the in vivo expression of an optimized uracil phosphoribosyltransferase (UPRT) enzyme. We demonstrate improved selectivity for metabolic incorporation of a modified nucleobase (5-vinyuracil) into nascent RNA, using a battery of tests. The selective incorporation of vinyl-U residues was demonstrated in 3xUPRT LM2 cells through validation with dot blot, qPCR, LC-MS/MS and microscopy analysis. We also report using this approach in a metastatic human breast cancer mouse model for profiling cell-specific nascent RNA.

Identifying Mechanisms of Resistance to DHODH Inhibition in Cancer

All transcriptionally active and rapidly dividing cells require high amounts of pyrimidines for DNA replication and repair and RNA synthesis. The only two known mechanisms by which cells can acquire pyrimidines are the de novo pyrimidine biosynthesis pathway and the pyrimidine salvage pathway. The rate-limiting enzyme in de novo synthesis is dihydroorotate dehydrogenase (DHODH), a critical metabolic enzyme that converts dihydroorotate (DHO) to orotate, which is then converted to the pyrimidine precursor uridine monophosphate (UMP). The rate-limiting enzyme in the salvage pathway is uridine-cytidine kinase 2 (UCK2), a ribonucleoside kinase that converts uridine to UMP and cytidine to cytidine monophosphate (CMP). Under physiologic conditions, salvage pathway substrates are limiting, and many cancer types, most notably acute myeloid leukemia (AML), are highly dependent on DHODH activity for proliferation and survival. Based on these observations, several DHODH inhibitors have been developed as anti-cancer therapeutics and have been tested clinically. Unfortunately, to date, these drugs have failed to show significant anti-cancer activity in early-phase clinical trials. In order to understand how leukemia cells that are highly sensitive to DHODH inhibitors can bypass their dependence on DHODH activity, we conducted positive-selection whole-genome CRISPR-activation drug resistance screens in TF-1 human AML cells using BAY 2402234, a highly potent and highly specific DHODH inhibitor. In preliminary studies, we found that 1 nM BAY 2402234 fully inhibits TF-1 cells whereas TF-1 cells that overexpress high levels of DHODH require 50 nM BAY 2402234 to achieve the same effect. Importantly, the effects of 1 nM and 50 nM BAY 2402234 are fully rescued by addition of uridine to the culture media, demonstrating that the anti-leukemic activity of BAY 2402234 is on-target at both drug doses. To perform the drug resistance screens, we stably expressed nuclease-deactivated Cas9 fused to a transcriptional activator (dCas9-VP64) in TF-1 cells and then infected the cells with the human whole-genome Calabrese sgRNA lentiviral library. We then passaged the cells in media containing vehicle control (DMSO), 1 nM or 50 nM BAY 2402234 for 18 days, collecting cells on day 0 and day 18 of drug treatment. Genomic DNA was then extracted and was subjected to next generation sequencing to assess the relative abundance of each sgRNA in each sample. We reasoned that activation of genes that enhance de novo pathway activity at the level of, or downstream of, DHODH would promote resistance to 1 nM BAY 2402234 but not 50 nM BAY 2402234, whereas activation of genes that act through de novo pathway-independent mechanisms would promote resistance to both 1 nM and 50 nM BAY 2402234. As predicted, sgRNAs targeting DHODH conferred resistance to 1 nM but not 50 nM BAY 2402234 whereas sgRNAs targeting UCK2 conferred resistance to both 1 and 50 nM BAY 2402234 (Figure 1). A number of other known salvage pathway genes scored in both screens, including uracil phosphoribosyltransferase (UPRT) and the nucleoside transporters SLC4A1 and SLC29A1. Unexpectedly, the screens also identified carbamoyl-phosphate synthetase 2/aspartate transcarbamoylase/dihydroorotase (CAD) as a potential mechanism of resistance to DHODH inhibition. CAD is a trifunctional multi-domain enzyme that acts upstream of DHODH, catalyzing the first three steps in de novopyrimidine biosynthesis. How upregulation of CAD activity can bypass the requirement for DHODH activity is not clear, although CAD is a very large enzyme complex about which very little is known besides its de novo pyrimidine synthesis activity. The screens also identified several genes that are not known to play a role in pyrimidine metabolism as potential resistance mechanisms. These include the serine-threonine kinase MARK3, the serine-threonine phosphatase CTDSPL2, and the RNA-binding protein NANOS2. Finally, the screens identified several genes whose upregulation sensitized cells to BAY 2402234, including the urokinase receptor PLAUR, the adaptor protein BCAR3, the scaffold protein SHOC2, and the deoxynucleoside phosphohydrolase SAMHD1. Studies are currently underway to validate the results of the CRISPRa screens and elucidate the mechanisms by which the candidate resistance genes help cells to bypass their requirement for DHODH activity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cryopreservation does not change the performance and characteristics of allogenic mesenchymal stem cells highly over-expressing a cytoplasmic therapeutic transgene for cancer treatment

BackgroundMesenchymal stem cells (MSCs) driven gene directed enzyme prodrug therapy is a promising approach to deliver therapeutic agents to target heterogenous solid tumours. To democratize such a therapy, cryopreservation along with cold chain transportation is an essential part of the logistical process and supply chain. Previously, we have successfully engineered MSCs by a non-viral DNA transfection approach for prolonged and exceptionally high expression of the fused transgene cytosine deaminase, uracil phosphoribosyl transferase and green fluorescent protein (CD::UPRT::GFP). The aim of this study was to determine the effects of cryopreservation of MSCs engineered to highly overexpress this cytoplasmic therapeutic transgene.MethodsModified MSCs were preserved in a commercially available, GMP-grade cryopreservative—CryoStor10 (CS10) for up to 11 months. Performance of frozen-modified MSCs was compared to freshly modified equivalents in vitro. Cancer killing potency was evaluated using four different cancer cell lines. Migratory potential was assessed using matrigel invasion assay and flow cytometric analysis for CXCR4 expression. Frozen-modified MSC was used to treat canine patients via intra-tumoral injections, or by intravenous infusion followed by a daily dose of 5-flucytosine (5FC).ResultsWe found that cryopreservation did not affect the transgene expression, cell viability, adhesion, phenotypic profile, and migration of gene modified canine adipose tissue derived MSCs. In the presence of 5FC, the thawed and freshly modified MSCs showed comparable cytotoxicity towards one canine and three human cancer cell lines in vitro. These cryopreserved cells were stored for about a year and then used to treat no-option-left canine patients with two different types of cancers and notably, the patients showed progression-free interval of more than 20 months, evidence of the effectiveness in treating spontaneously occurring cancers.ConclusionThis study supports the use of cryopreserved, off-the-shelf transiently transfected MSCs for cancer treatment.

Metabolic labeling of cardiomyocyte-derived small extracellular-vesicle (sEV) miRNAs identifies miR-208a in cardiac regulation of lung gene expression.

Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) converts 4‐thiouracil (4TUc) into 4‐thiouridine (4TUd), which is incorporated into nascent RNAs and can be biotinylated, then labelled with streptavidin conjugates or isolated via streptavidin‐affinity methods. Here, we generated mice that expressed T. gondii UPRT only in cardiomyocytes (CMUPRT mice) and tested our hypothesis that CM‐derived miRNAs (CMmiRs) are transferred into remote organs after myocardial infarction (MI) by small extracellular vesicles (sEV) that are released from the heart into the peripheral blood (PBsEV). We found that 4TUd was incorporated with high specificity and sensitivity into RNAs isolated from the hearts and PBsEV of CMUPRT mice 6 h after 4TUc injection. In PBsEV, 4TUd was incorporated into CM‐specific/enriched miRs including miR‐208a, but not into miRs with other organ or tissue‐type specificities. 4TUd‐labelled miR208a was also present in lung tissues, especially lung endothelial cells (ECs), and CM‐derived miR‐208a (CMmiR‐208a) levels peaked 12 h after experimentally induced MI in PBsEV and 24 h after MI in the lung. Notably, miR‐208a is expressed from intron 29 of α myosin heavy chain (αMHC), but αMHC transcripts were nearly undetectable in the lung. When PBsEV from mice that underwent MI (MI‐PBsEV) or sham surgery (Sham‐PBsEV) were injected into intact mice, the expression of Tmbim6 and NLK, which are suppressed by miR‐208a and cooperatively regulate inflammation via the NF‐κB pathway, was lower in the lungs of MI‐PBsEV–treated animals than the lungs of animals treated with Sham‐PBsEV or saline. In MI mice, Tmbim6 and NLK were downregulated, whereas endothelial adhesion molecules and pro‐inflammatory cells were upregulated in the lung; these changes were significantly attenuated when the mice were treated with miR‐208a antagomirs prior to MI surgery. Thus, CMUPRT mice enables us to track PBsEV‐mediated transport of CMmiRs and identify an miR‐208a‐mediated mechanism by which myocardial injury alters the expression of genes and inflammatory response in the lung.

Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides.

The discovery of previously unknown functional roles of RNA in biological systems has led to increased interest in revealing novel RNA molecules as therapeutic targets and the development of tools to better understand the role of RNA in cells. RNA metabolic labeling broadens the scope of studying RNA by incorporating of unnatural nucleobases and nucleosides with bioorthogonal handles that can be utilized for chemical modification of newly synthesized cellular RNA. Such labeling of RNA provides access to applications including measurement of the rates of synthesis and decay of RNA, cellular imaging for RNA localization, and selective enrichment of nascent RNA from the total RNA pool. Several unnatural nucleosides and nucleobases have been shown to be incorporated into RNA by endogenous RNA synthesis machinery of the cells. RNA metabolic labeling can also be performed in a cell-specific manner, where only cells expressing an essential enzyme incorporate the unnatural nucleobase into their RNA. Although several discoveries have been enabled by the current RNA metabolic labeling methods, some key challenges still exist: (i) toxicity of unnatural analogues, (ii) lack of RNA-compatible conjugation chemistries, and (iii) background incorporation of modified analogues in cell-specific RNA metabolic labeling. In this Account, we showcase work done in our laboratory to overcome these challenges faced by RNA metabolic labeling.To begin, we discuss the cellular pathways that have been utilized to perform RNA metabolic labeling and study the interaction between nucleosides and nucleoside kinases. Then we discuss the use of vinyl nucleosides for metabolic labeling and demonstrate the low toxicity of 5-vinyluridine (5-VUrd) compared to other widely used nucleosides. Next, we discuss cell-specific RNA metabolic labeling with unnatural nucleobases, which requires the expression of a specific phosphoribosyl transferase (PRT) enzyme for incorporation of the nucleobase into RNA. In the course of this work, we discovered the enzyme uridine monophosphate synthase (UMPS), which is responsible for nonspecific labeling with modified uracil nucleobases. We were able to overcome this background labeling by discovering a mutant uracil PRT (UPRT) that demonstrates highly specific RNA metabolic labeling with 5-vinyluracil (5-VU). Furthermore, we discuss the optimization of inverse-electron-demand Diels-Alder (IEDDA) reactions for performing chemical modification of vinyl nucleosides to achieve covalent conjugation of RNA without transcript degradation. Finally, we highlight our latest endeavor: the development of mutually orthogonal chemical reactions for selective labeling of 5-VUrd and 2-vinyladenosine (2-VAdo), which allows for potential use of multiple vinyl nucleosides for simultaneous investigation of multiple cellular processes involving RNA. We hope that our methods and discoveries encourage scientists studying biological systems to include RNA metabolic labeling in their toolkit for studying RNA and its role in biological systems.

Snapshots of nascent RNA reveal cell- and stimulus-specific responses to acute kidney injury.

The current strategy to detect acute injury of kidney tubular cells relies on changes in serum levels of creatinine. Yet serum creatinine (sCr) is a marker of both functional and pathological processes and does not adequately assay tubular injury. In addition, sCr may require days to reach diagnostic thresholds, yet tubular cells respond with programs of damage and repair within minutes or hours. To detect acute responses to clinically relevant stimuli, we created mice expressing Rosa26-floxed-stop uracil phosphoribosyltransferase (Uprt) and inoculated 4-thiouracil (4-TU) to tag nascent RNA at selected time points. Cre-driven 4-TU–tagged RNA was isolated from intact kidneys and demonstrated that volume depletion and ischemia induced different genetic programs in collecting ducts and intercalated cells. Even lineage-related cell types expressed different genes in response to the 2 stressors. TU tagging also demonstrated the transient nature of the responses. Because we placed Uprt in the ubiquitously active Rosa26 locus, nascent RNAs from many cell types can be tagged in vivo and their roles interrogated under various conditions. In short, 4-TU labeling identifies stimulus-specific, cell-specific, and time-dependent acute responses that are otherwise difficult to detect with other technologies and are entirely obscured when sCr is the sole metric of kidney damage.

A Three-Dimensional Organoid Model of Primary Breast Cancer to Investigate the Effects of Oncolytic Virotherapy

Background: Although several oncolytic viruses have already been tested in early-stage clinical studies of breast cancer, there is still an urgent need to develop patient-derived experimental systems that mimic the response of breast cancer to oncolytic agents in preparation of testing different oncolytic viruses in clinical trials. We addressed this need by developing a protocol to study the effects of oncolytic viruses in stable organoid cell cultures derived from breast cancer tissue. Methods: We used an established three-dimensional organoid model derived from tissue of 10 patients with primary breast cancer. We developed an experimental protocol for infecting organoid cultures with oncolytic viruses and compared the oncolytic effects of a measles vaccine virus (MeV) and a vaccinia virus (GLV) genetically engineered to express either green fluorescent protein (MeV-GFP) and red fluorescent protein (GLV-0b347), respectively, or a suicide gene encoding a fusion of cytosine deaminase with uracil phosphoribosyltransferase (MeV-SCD and GLV-1h94, respectively), thereby enabling enzymatic conversion of the prodrug 5-fluorocytosine (5-FC) into cytotoxic compounds 5-fluorouracil (5-FU) and 5-fluorouridine monophosphate (5-FUMP). Results: The method demonstrated that all oncolytic viruses significantly inhibited cell viability in organoid cultures derived from breast cancer tissue. The oncolytic effects of the oncolytic viruses expressing suicide genes (MeV-SCD and GLV-1h94) were further enhanced by virus-triggered conversion of the prodrug 5-FC to toxic 5-FU and toxic 5-FUMP. Conclusions: We were able to develop a protocol to assess the effects of two different types of oncolytic viruses in stable organoid cell cultures derived from breast cancer tissue. The greatest oncolytic effects were observed when the oncolytic viruses were engineered to express a suicide gene (MeV-SCD and GLV-1h94) in the presence of the prodrug 5-FC. The model therefore provides a promising in vitro method to help further testing and engineering of new generations of virotherapeutic vectors for in vivo use.

Gene-Directed Enzyme/Prodrug Therapy of Rat Brain Tumor Mediated by Human Mesenchymal Stem Cell Suicide Gene Extracellular Vesicles In Vitro and In Vivo.

Simple SummaryExtracellular vesicles— exosomes—secreted by human mesenchymal stem/stromal cells are able to cross the blood–brain barrier and internalize glioblastoma cells. We prepared exosomes possessing a gene message, the product of which is able to convert nontoxic 5-fluorocytosine to cytotoxic drug 5-fluorouracil. Such therapeutic exosomes administered intranasally, intraperitoneally, or subcutaneously to rats bearing intracerebral glioblastoma cells inhibited their growth. The treatment cured a significant number of animals.MSC-driven, gene-directed enzyme prodrug therapy (GDEPT) mediated by extracellular vesicles (EV) represents a new paradigm—cell-free GDEPT tumor therapy. In this study, we tested the efficacy of yeast cytosine deaminase::uracilphosphoribosyl transferase (yCD::UPRT-MSC)-exosomes, in the form of conditioned medium (CM) to inhibit the growth of C6 glioblastoma cells both in vitro and in vivo. MSCs isolated from human adipose tissue, umbilical cord, or dental pulp engineered to express the yCD::UPRT gene secreted yCD::UPRT-MSC-exosomes that in the presence of the prodrug 5-fluorocytosine (5-FC), inhibited the growth of rat C6 glioblastoma cells and human primary glioblastoma cells in vitro in a dose-dependent manner. CM from these cells injected repeatedly either intraperitoneally (i.p.) or subcutaneously (s.c.), applied intranasally (i.n.), or infused continuously by an ALZET osmotic pump, inhibited the growth of cerebral C6 glioblastomas in rats. A significant number of rats were cured when CM containing yCD::UPRT-MSC-exosomes conjugated with 5-FC was repeatedly injected i.p. or applied i.n. Cured rats were subsequently resistant to challenges with higher doses of C6 cells. Our data have shown that cell-free GDEPT tumor therapy mediated by the yCD::UPRT-MSC suicide gene EVs for high-grade glioblastomas represents a safer and more practical approach that is worthy of further investigation.

Uniport, Not Proton-Symport, in a Non-Mammalian SLC23 Transporter

SLC23 family members are transporters of either nucleobases or ascorbate. While the mammalian SLC23 ascorbate transporters are sodium-coupled, the non-mammalian nucleobase transporters have been proposed, but not formally shown, to be proton-coupled symporters. This assignment is exclusively based on in vivo transport assays using protonophores. Here, by establishing the first in vitro transport assay for this protein family, we demonstrate that a representative member of the SLC23 nucleobase transporters operates as a uniporter instead. We explain these conflicting assignments by identifying a critical role of uracil phosphoribosyltransferase, the enzyme converting uracil to UMP, in driving uracil uptake in vivo. Detailed characterization of uracil phosphoribosyltransferase reveals that the sharp reduction of uracil uptake in whole cells in presence of protonophores is caused by acidification-induced enzyme inactivation. The SLC23 family therefore consists of both uniporters and symporters in line with the structurally related SLC4 and SLC26 families that have previously been demonstrated to accommodate both transport modes as well.

Taylor-made production of pyrimidine nucleoside-5′-monophosphate analogues by highly stabilized mutant uracil phosphoribosyltransferase from Toxoplasma gondii

Nowadays, enzymatic synthesis of nucleotides is an efficient and sustainable alternative to chemical methodologies. In this regard, after the biochemical characterization of wild-type and mutant uracil phosphoribosyltransferases from Toxoplasma gondii (TgUPRT, TgUPRT2, and TgUPRT3), TgUPRT2 was selected as the optimal candidate (69.5 IU mg−1, UMP synthesis) for structure-guided immobilization onto Ni2+ chelate (MNiUPRT2) and onto glutaraldehyde-activated microparticles (MGlUPRT2). Among resulting derivatives, MNiUPRT23 (6127 IU g−1biocat; 92% retained activity; 3–5 fold enhanced stability at 50–60 °C) and MGlUPRT2N (3711 IU g−1biocat; 27% retained activity; 8–20 fold enhanced stability at 50–60 °C) displayed the best operability. Moreover, the enzymatic synthesis of different pyrimidine NMPs was performed. Finally, the reusability of both derivatives in 5-FUMP synthesis (MNiUPRT23, 80% retained activity after 7 cycles, 5 min; MGlUPRT2N, 70% retained activity after 10 cycles, 20 min) was carried out at short times.

Single-plasmid systems based on CRISPR-Cas9 for gene editing in Lactococcus lactis

Lactococcus lactis is a food-grade lactic acid bacterial species that is widely used in food and medical industries. Due to its relatively small genome and simple metabolism, L. lactis is commonly engineered to produce large quantities of recombinant proteins. The most common single-gene knockout strategy in L. lactis involves RecA-dependent homologous double-crossover recombination, which is relatively time-consuming and laborious. In this study, a precise and efficient genome-editing plasmid for L. lactis NZ9000 genome engineering, pLL, was established based on clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. By studying the effects of different single guide RNA (sgRNA) promoters, the efficiency of gene deletion was optimized. For LLNZ_02045 (ldh), gene deletion efficiency of up to 50% was achieved. Effective sequential gene deletion of LLNZ_11240 (upp) and LLNZ_04580 (upp1) was also demonstrated using this tool. Additionally, the gene that encodes for uracil phosphoribosyltransferase was identified using this system. Similar robust gene deletion efficiencies of sgRNA that targeted different regions of a single gene suggested that gene deletion was not affected by the location of sgRNA binding. Thus, our study established a new gene-editing tool that may allow further investigation and understanding of the L. lactis NZ9000 genome.

Computational Approach to study the effect of point mutations in the development of antifungal resistance to Azoles and Flucytosine Drugs in Candida auris

Background: Candida auris is associated with invasive and severe candidemia, multi-drug resistance and high mortalities. Azoles and Flucytosine are commonly used antifungal drugs. Lanosterol alpha-demethylase (ERG11), Uracil phosphoribosyl transferase (FUR1) are two principal proteins involved in ergosterol biosynthesis and pyrimidine metabolism. However, crystal structures of these proteins from C. auris have not yet been established. We constructed structural model of ERG11 and FUR1 proteins for South-African Clade using homology modelling, molecular docking and molecular dynamics simulations. To investigate how point mutations affect drug interaction, we used the same methods on ERG11 mutants (Y132F, K143R) and FUR1 mutants (F211I). Methodology: Homology modelling was used to construct 3D structure of proteins. Reliability of models was analysed by using validation tools. The drug interaction in wild and mutant variants was studied using molecular docking, and binding energy was calculated. Finally, we investigated structural significance of point-mutation between two variants of FUR1 through MD Simulation. Result: Structural models of ERG11 and FUR1 were compared based on binding energy and hydrogen bonding. Few azole compounds showed no effect of mutation on interaction. Further, it was found that binding affinity for 5-fluorocytosine decreases in the mutant variant of FUR1. MD Simulation of wild variant FUR1-5FC complex showed stabilisation till 7ns while mutated complex was stable for 4.5ns. Conclusion: C. auris resistance to antifungal drugs poses a significant risk to public health. The study sheds light on how drug interactions are influenced by mutations and aids in the development of antifungal drugs.

Development of LC-HRMS methods for evaluation of metabolic conversion of 5-fluorocytosine at GDEPT procedure

Gene-directed enzyme/prodrug therapy represents one of the experimental treatment approaches. The system based on conversion of nontoxic prodrug 5-fluorocytosine to chemotherapeutic 5-fluorouracil by cytosine deaminase or fusion cytosine deaminase::uracil phosphoribosyl transferase belongs to the most frequently used. The detailed analysis of 5-fluorocytosine, 5-fluorouracil and its metabolites enables to understand various responses of tumour cells to treatment as well as mechanisms of resistance.A fast, sensitive and accurate methods based on liquid chromatography with high-resolution mass spectrometry (LC-HRMS) for the identification and quantification of 5-fluorocytosine, 5-fluorouracil and its major metabolites were developed. Two different hybrid high-resolution mass spectrometers sufficient for study of metabolic pathways were used. The LC-ESI IT-TOF MS method was successfully used for identification of 5-fluorocytosine, 5-fluorouracil and its metabolites in complex biological matrices (mesenchymal stromal cells and tumour cells media) and for confirmation of the metabolic conversion of 5-fluorocytosine even in chemoresistant tumour cells media samples. For quantification, the LC-HESI QExactive MS method was developed and validated. The developed method demonstrated a very good linear range for 5-fluorocytosine from 1 ng/mL to 1000 ng/mL and for its major metabolites from 5 ng/mL to 1000 ng/mL. The limits of detection and limits of quantification ranged from 1.1 to 26 ng/mL and from 3.6 to 87 ng/mL, respectively.Both developed methods confirmed the ability of gene-directed enzyme prodrug therapy to metabolically convert 5-fluorocytosine to 5-fluorouracil and its major metabolites in real samples of tumour cell media and mesenchymal stromal cells.