Kinetic studies on the chymotrypsin Aα-catalyzed hydrolysis of a series of N-acetyl-l-phenylalanyl peptides

A series of N-acetyl-l-phenylalanyl peptides of general formula Ac-Phe-(Gly)_n-NH₂ (n = 0–2) has been synthesized to study the effect of leaving group chain length on the efficiency of chymotrypsin A_α amidase and peptidase activities. The effect upon catalysis of hydrophobic side chains on the leaving group was investigated using similar substrates with one of the glycine residues selectively substituted by an alanine residue as in AcPheAlaNH₂, AcPheAlaGlyNH₂, and AcPheGlyAlaNH₂. Values of k_cat and K_m have been obtained from kinetic measurements at pH 8.00 and 25 °C. The results are shown to be consistent with binding schemes postulated from published model building studies. The catalytic reactions were studied over a range of temperature (15–35 °C) and in each case the Arrhenius law was obeyed. It was thus possible to obtain meaningful values for the thermodynamic functions of activation for the acylation step of the catalytic reaction. The results are shown to confirm the findings of postulated binding schemes but indicate that conclusions drawn from kinetic measurements at a single temperature may sometimes be misleading.     

Characterization of the Prostate-Specific Antigen (PSA) Catalytic Mechanism: A Pre-Steady-State and Steady-State Study

Prostate-specific antigen (PSA), an enzyme of 30 kDa grouped in the kallikrein family is synthesized to high levels by normal and malignant prostate epithelial cells. Therefore, it is the main biomarker currently used for early diagnosis of prostate cancer. Here, presteady-state and steady-state kinetics of the PSA-catalyzed hydrolysis of the fluorogenic substrate Mu-His-Ser-Ser-Lys-Leu-Gln-AMC (spanning from pH 6.5 to pH 9.0, at 37.0°C) are reported. Steady-state kinetics display at every pH value a peculiar feature, represented by an initial “burst” phase of the fluorescence signal before steady-state conditions are taking place. This behavior, which has been already observed in other members of the kallikrein family, suggests the occurrence of a proteolytic mechanism wherefore the acylation step is faster than the deacylation process. This feature allows to detect the acyl intermediate, where the newly formed C-terminal carboxylic acid of the cleaved substrate forms an ester bond with the -OH group of the Ser195 catalytic residue, whereas the AMC product has been already released. Therefore, the pH-dependence of the two enzymatic steps (i.e., acylation and deacylation) has been separately characterized, allowing the determination of pK_a values. On this basis, possible residues are tentatively identified in PSA, which might regulate these two steps by interacting with the two portions of the substrate.     

Enhanced acylation activity of esterase BioH from Escherichia coli by directed evolution towards improved hydrolysis activity

Esterase BioH is a critical enzyme for Biotin synthesis in Escherichia coli, which has been previously found to be active in the acylation of secondary alcohols and amines. Directed evolution towards improved acylation activity requires a high-throughput screening method. The aim of this study is to explore whether the acylation activity of BioH can be improved by directed evolution of its hydrolysis activity. A colorimetric method based on p-nitrophenyl butyrate hydrolysis was adopted in the high-throughput determination of hydrolysis activity. The wild-type BioH showed a hydrolysis activity of 18 U/mg, and the specific activities for α-phenylethanol and α-phenylethylamine acylation were 12.8 U/mg and 3.5 U/mg, respectively. After two rounds of directed evolution, seven mutants with improved hydrolysis activity were obtained, among which, K213E, Q70L/M170T and M197L/K213E also showed improvement in acylation activity. To further improve the acylation activity, site mutations were generated in different combinations at the four hot spots Q70L, M170T, M197L and K213E. Among the resulting mutants, Q70L/M197L/K213E showed the highest activity in α-phenylethylamine acylation with a 120% improvement, while Q70L/K213E had the highest α-phenylethanol acylation activity, which was improved by 70%. The results demonstrated that directed evolution of the hydrolysis activity might be a possible approach to improve the acylation activity of the esterase BioH.     

Penicillin G acylase catalyzed acylation of 7-ACA in aqueous two-phase systems using kinetically and thermodynamically controlled strategies: improved enzymatic synthesis of 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Δ3-cephem-4-carboxylic acid

Penicillin G acylase (PGA) catalyzed acylation of 7-aminocephalosporanic acid (7-ACA) with R-mandelic acid and its derivatives gives 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Δ³-cephem-4-carboxylic acid. This compound is a useful intermediate for the synthesis of some 3′-functionalized cephalosporins. However, acylations catalyzed by PGA isolated from Escherichia coli give poor results both considering a kinetical or a thermodynamical approach. In order to improve this enzymatic acylation, polyethylene glycol (PEG 600)-ammonium sulphate aqueous two-phase systems have been studied with the aim to have, during the reaction, a continuous extraction of the acylation product outside of the enzyme environment (the ammonium sulphate phase). This strategy shifts the equilibrium in the thermodynamically controlled synthesis and prevents the hydrolysis of the synthesized antibiotic in the kinetically controlled synthesis. The best results were achieved using PEG 600 (80% in water) equilibrated with 4 M ammonium sulphate. In these conditions, the acylation product was completely partitioned in the PEG phase (K > 200), whereas the substrates maintained a suitable concentration in the enzyme environment. Both in the kinetic (88% yield) and the thermodynamic (75% yield) processes, the results obtained were sensitively improved in comparison with those achieved working in homogeneous solution (phosphate buffer). Using R-mandelic acid methyl ester, the yield increased from 65% (monophasic system) to 88%. The PEG solution, without isolation of the acylation product, was successfully used for the synthesis of Cefamandole and Cefonicid.     

Mechanism of heparin acceleration of tissue inhibitor of metalloproteases-1 (TIMP-1) degradation by the human neutrophil elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k ₂ = 21±1 s⁻¹) was much higher than the HNE deacylation step (k ₃ = 0.57±0.05 s⁻¹). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k ₁ 2.4-fold and reducing k ₋₁ 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k ₂ value, whereas the k ₃ value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs.     

Identification of acylation products in SHAPE chemistry

SHAPE chemistry (selective 2′-hydroxyl acylation analyzed by primer extension) has been developed to specifically target flexible nucleotides (often unpaired nucleotides) independently to their purine or pyrimidine nature for RNA secondary structure determination. However, to the best of our knowledge, the structure of 2′-O-acylation products has never been confirmed by NMR or X-ray data. We have realized the acylation reactions between cNMP and NMIA under SHAPE chemistry conditions and identified the acylation products using standard NMR spectroscopy and LC–MS/MS experiments. For cAMP and cGMP, the major acylation product is the 2′-O-acylated compound (>99%). A trace amount of N-acylated cAMP has also been identified by LC–UV–MS². While for cCMP, the isolated acylation products are composed of 96% of 2′-O-acylated, 4% of N,O-diacylated, and trace amount of N-acylated compounds. In addition, the characterization of the major 2′-O-acylated compound by NMR showed slight differences in the conformation of the acylated sugar between the three cyclic nucleotides. This interesting result should be useful to explain some unexpected reactivity of the SHAPE chemistry.     

ZNF76 predicts prognosis and response to platinum chemotherapy in human ovarian cancer

Ovarian cancer (OV) is the most lethal gynecologic malignancy. One major reason of the high mortality of the disease is due to platinum-based chemotherapy resistance. Increasing evidence reveal the important biological functions and clinical significance of zinc finger proteins (ZNFs) in OV. In the present study, the relationship between the zinc finger protein 76 (ZNF76) and clinical outcome and platinum resistance in patients with OV was explored. We further analyzed ZNF76 expression via multiple gene expression databases and identified its functional networks using cBioPortal. RT-qPCR and IHC assay shown that the ZNF76 mRNA and protein expression were significantly lower in OV tumor than that in normal ovary tissues. A strong relationship between ZNF76 expression and platinum resistance was determined in patients with OV. The low expression of ZNF76 was associated with worse survival in OV. Multivariable analysis showed that the low expression of ZNF76 was an independent factor predicting poor outcome in OV. The prognosis value of ZNF76 in pan-cancer was validated from multiple cohorts using the PrognoScan database and GEPIA 2. A gene-clinical nomogram was constructed by multivariate cox regression analysis, combined with clinical characterization and ZNF76 expression in TCGA. Functional network analysis suggested that ZNF76 was involved in several biology progressions which associated with OV. Ten hub genes (CDC5L, DHX16, SNRPC, LSM2, CUL7, PFDN6, VARS, HSD17B8, PPIL1, and RGL2) were identified as positively associated with the expression of ZNF76 in OV. In conclusion, ZNF76 may serve as a promising prognostic-related biomarker and predict the response to platinum in OV patients.     

Catalytic action of comicelles containing imidazolyl and hydroxyl groups in the hydrolysis of enantiomeric esters

The rate constants for hydrolysis of the enantiomers of amino acid p-nitrophenyl esters catalyzed by bifunctional comicellar catalysts containing the imidazolyl and hydroxyl groups have been determined at pH 7.30, 0.02 m phosphate buffer, and 25°C. The kinetic analysis suggests a reaction scheme which involves acylation followed by deacylation at the imidazolyl group. Although no appreciable cooperative catalytic efficiencies are observed between the bifunctional groups in the acylation step, it is found that the deacylation rates are thus accelerated by surfactant hydroxyl groups, and some of the stereoselective acyl transfer reaction occurs from the imidazolyl to the hydroxyl group in optically active comicellar systems.     

Chiral discrimination using a quartz crystal microbalance and comparison with gas chromatographic retention data

Quartz crystal microbalances (QCMB) have been constructed using 10 MHz AT cut quartz crystals coated with heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, heptakis(6-O-methyl-2,3-di-O-pentyl)-β-cyclodextrin, and octakis(6-O-methyl-2,3-di-O-pentyl)-γ-cyclodextrin as 50% and 20% (w/w) solutions in OV1701. The reduction in frequency seen on exposure of each coated QCMB to pure enantiomeric forms of α- and β-pinene and cis- and trans-pinane show that statistically significant (P = 0.05, n = 7) differences are observed between the enantiomeric pairs. The apparent preferential binding shown by the QCMB for enanciomers of α- and β-pinene and cis- and trans-pinane have been compared with the elution order observed on the corresponding gas chromatographic stationary phase. The magnitude of the observed separation factor (calculated as the ratio of the OV1701 normalised frequency shift) is seen to be dependent upon the chiral stationary phase concentration. These results indicate that on-line determination of enantiomeric excess and concentration of certain monoterpenes is possible at room temperature using QCMB in conjunction with chiral gas chromatographic stationary phases. Chirality 9:225–232, 1997. © 1997 Wiley-Liss, Inc.     

A rate-limiting step of enteropeptidase hydrolysis

It has been shown for the first time that deacylation is the rate-limiting step in the enteropeptidase-catalyzed hydrolysis of highly effective oligopeptide substrates containing four Asp residues in positions P2–P5. On the other hand, the rate-limiting step in the hydrolysis of low-efficiency peptide substrates containing less than four Asp or Glu residues in positions P2–P5 is acylation, as it has previously been suggested for all amide and peptide substrates of serine proteases on the basis of classical works of Bender et al. The method of introduction of an additional nucleophile or another effector that selectively affects the deacylation step was used to determine the rate-limiting step in the enteropeptidase hydrolysis of N ^α-benzyloxycarbonyl-L-lysine thiobenzyl ester, the highly efficient amide substrate GlyAsp₄-Lys β-naphthyl amide, and the low-efficiency peptide substrate VLSAADK-GNVKAAWG (where a hyphen denotes the hydrolysis site).     

Anteroventrally localized activity in the optic vesicle plays a crucial role in the optic development

The vertebrate eye develops from the optic vesicle (OV), a laterally protrusive structure of the forebrain, by a coordinated interaction with surrounding tissues. The OV then invaginates to form an optic cup, and the lens placode develops to the lens vesicle at the same time. These aspects in the early stage characterize vertebrate eye formation and are controlled by appropriate dorsal-ventral coordination. In the present study, we performed surgical manipulation in the chick OV to remove either the dorsal or ventral half and examined the development of the remaining OV. The results show that the dorsal and ventral halves of the OV have a clearly different developmental pattern. When the dorsal half was removed, the remaining ventral OV developed into an entire eye, while the dorsal OV developed to a pigmented vesicle consisting of retinal pigmented epithelium alone. These results indicate that the ventral part of the OV retains the potency to develop the entire eye structure and plays an essential role in proper eye development. In subsequent manipulations of early chick embryos, it was found that only the anterior ventral quadrant of the OV has the potential to develop the entire eye and that no other part of the OV has a similar activity. Fgf8 expression was localized in this portion and no Fgf8 expression was observed within the OV when the ventral OV was removed. These results suggest that the anterior ventral portion of the OV plays a crucial role in the proper development of the eye, possibly generating the dorsal-ventral gradients of signal proteins within the eye primordium.     

The antiproliferative effect of hexadecylphosphocholine toward HL60 cells is prevented by exogenous lysophosphatidylcholine

The mechanisms that account for the anti-proliferative properties of the biologically active lysophospholipid analog hexadecylphosphocholine (HexPC) were investigated in HL60 cells. HexPC inhibited the incorporation of choline into phosphatidylcholine and the pattern of accumulation of soluble choline-derived metabolites pinpointed CTP:phosphocholine cytidylyltransferase (CT) as the inhibited step in vivo. HexPC also inhibited recombinant CT in vitro. HexPC treatment led to accumulation of cells in G₂/M phase, triggered DNA fragmentation and caused morphological changes associated with apoptosis. The supplementation of HexPC-treated cells with exogenous lysophosphatidylcholine (LPC) completely reversed the cytotoxic effects of HexPC and restored HL60 cell proliferation in the presence of the drug. LPC provided an alternate pathway for phosphatidylcholine synthesis via the acylation of exogenous LPC. This result contrasted with the response of HL60 cells to 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃) where LPC overcame the cytotoxic effects but did not support continued cell proliferation. Morphological integrity, DNA stability and cell viability were maintained in cells treated with LPC plus either antineoplastic agent. Thus the inhibition of phosphatidylcholine biosynthesis at the CT step accounts for the cytotoxicity of both HexPC and ET-18-OCH₃ which is overridden by providing an alternate pathway for phosphatidylcholine synthesis via the acylation of exogenous LPC.     

Sonochemical enzyme-catalyzed regioselective acylation of flavonoid glycosides

This work compares a highly efficient and alternative method of sonication-assisted lipase catalyzed acylation of quercetin-3-O-glucoside and phloretin-2′-glucoside, using Candida antarctica lipase B (Novozyme 435^®), with a range of fatty acids. In this study, sonication-assisted irradiation coupled with stirring has been found to be more efficient and economical than conventional reaction conditions. Sonication-assisted acylation accelerated the reactions and reduced the time required by 4–5 folds.     

Distribution and Abundance of Opisthorchis viverrini Metacercariae in Cyprinid Fish in Northeastern Thailand

To increase public health awareness for prevention of opisthorchiasis caused by eating raw freshwater fish, the distribution and abundance of Opisthorchis viverrini metacercariae (OV MC) was investigated in freshwater fish obtained from 20 provinces in northeastern Thailand between April 2011 and February 2012. A cross-sectional survey was conducted on 12,890 fish consisting of 13 species randomly caught from 26 rivers, 10 dams, and 38 ponds/lakes. Fish, were collected in each of the rainy and winter seasons from each province. Fish were identified, counted, weighed, and digested using pepsin-HCl. Samples were examined for OV MC by a sedimentation method, and metacercariae were identified under a stereomicroscope. OV MC were found in 6 species of fish; i.e., Cyclocheilichthys armatus, Puntius orphoides, Hampala dispar, Henicorhynchus siamensis, Osteochilus hasselti, and Puntioplites proctozysron from localities in 13 provinces. Among the sites where OV MC-infected fish were found, 70.0% were dams, 23.7% were ponds/lakes, and 7.7% were rivers. The mean intensity of OV MC ranged from 0.01 to 6.5 cysts per fish (or 1.3-287.5 cysts per kg of fish). A high mean intensity of OV MC per fish (>3 cysts) was found in 5 provinces: Amnat Charoen (6.5 cysts), Nakhon Phanom (4.3), Mukdahan (4.1), Khon Kaen, (3.5) and Si Sa Ket (3.4). In conclusion, OV MC are prevalent in natural cyprinid fish, with the infection rate varying according to fish species and habitats.     

Advances in the Diagnosis of Human Opisthorchiasis: Development of Opisthorchis viverrini Antigen Detection in Urine

Background

Many strategies to control opisthorchiasis have been employed in Thailand, but not in the other neighbouring countries. Specific control methods include mass drug administration (MDA) and health education to reduce raw fish consumption. These control efforts have greatly shifted the epidemiology of Opisthorchis viverrini (OV) infection over the last decade from presenting as densely concentrated "heavy" infections in single villages to widespread "light" OV infections distributed over wide geographical areas. Currently, the "gold standard" detection method for OV infection is formalin ethyl-acetate concentration technique (FECT), which has limited diagnostic sensitivity and diagnostic specificity for light OV infections, with OV eggs often confused with eggs of minute intestinal flukes (MIFs) in feces. In this study, we developed and evaluated the diagnostic performance of a monoclonal antibody-based enzyme-linked immunosorbent assay for the measurement of OV excretory-secretory (ES) antigens in urine (urine OV-ES assay) for the diagnosis of opisthorchiasis compared to the gold standard detection FECT method.

Methodology

We tested several methods for pre-treating urine samples prior to testing the diagnostic performance of the urine OV-ES assay. Using trichloroacetic acid (TCA) pre-treated urine, we compared detection and quantification of OV infection using the urine OV-ES assay versus FECT in OV-endemic areas in Northeastern Thailand. Receiver operating characteristic (ROC) curves were used to determine the diagnostic sensitivity and specificity of the urine OV-ES assay using TCA pre-treated urine, and to establish diagnostic positivity thresholds. The Positive Predictive Value as well as the likelihood of obtaining a positive test result (LR+) or a negative test result (LR-) were calculated for the established diagnostic positivity threshold. Diagnostic risks (Odds Ratios) were estimated using logistic regression.

Results

When urine samples were pre-treated with TCA prior to use in the urine OV-ES assay, the analytical sensitivity was significantly improved. Using TCA pre-treatment of urine, the urine OV-ES assay had a limit of detection (LoD) of 39 ng/ml compared to the LoD of 52 ng/mL reported for coprological antigen detection methods. Similarly, the urine OV-ES assay correlated significantly with intensity of OV infection as measured by FECT. The urine OV-ES assay was also able to detect 28 individuals as positive from the 63 (44.4%) individuals previously determined to be negative using FECT. The likelihood of a positive diagnosis of OV infection by urine OV-ES assay increased significantly with the intensity of OV infection as determined by FECT. With reference to FECT, the sensitivity and specificity of the urine OV-ES assay was 81% and 70%, respectively.

Conclusion

The detection of OV-infection by the urine OV-ES assay showed much greater diagnostic sensitivity and diagnostic specificity than the current "gold standard" FECT method for the detection and quantification of OV infection. Due to its ease-of-use, and noninvasive sample collection (urine), the urine OV-ES assay offers the potential to revolutionize the diagnosis of liver fluke infection and provide an effective tool for control and elimination of these tumorigenic parasites.     

Purification of intracellular enzymes from whole bacterial cells using reversed micelles

A new, rapid pre-chromatography isolation procedure for intracellular enzymes from whole bacterial cells has been developed using reversed micelles. The method involves two relatively simple steps. In the first step, bacterial cells are disintegrated by the surfactants in the reversed micellar medium, and in the second step the liberated enzymes are extracted from the reversed micellar phase into an aqueous phase. The feasibility of using reversed micelles as a bioseparation tool has been demonstrated by following the activities and recoveries of three different dehydrogenases from Azotobacter vinelandii.     

Co-expression pattern of dopamine beta-hydroxylase (DβH) and neuropeptide Y (NPY) within sympathetic innervation of ovary and umbilical cord of the European bison (Bison bonasus L.)

Co-expression of dopamine β-hydroxylase (DβH) and neuropeptide Y (NPY) has never been examined in ovary (OV) and umbilical cord (UC) of the European bison (Eb), the endangered wild species. The OV and UC samples were harvested from seasonally eliminated Eb females (45–120 days post coitum). Frozen histological sections were examined by double fluorescent immunohistochemistry (dF-IHC), using the primary mouse anti-DβH monoclonals and rabbit anti-NPY polyclonals and then the immunocomplexes were visualized with FITC and CY3 fluorophores, respectively. Numerous DβH immunoreactive nerve fibers (DβH-IRs) and a little less frequent NPY-IRs were found in the bundle-like structures, innervating mainly perivascular regions of the OV. The NPY-IRs constantly co-expressed DβH, while some DβH-IRs did not express NPY. This specific pattern of innervation was observed both in the stromal and cortical regions of the OV. The simultaneous co-expression of DβH and NPY were also detected in the UC, in which specific single or bundle-like structures ran along the smooth muscles of blood vessels. The spatial-specific co-expression of DβH and NPY in OV and UC, may suggest that these markers are involved in the control of vascularization that regulates nourishing blood circulation required for proper pregnancy maintenance and efficient embryo/fetus development in the Eb.     

Risk factors for cholangiocarcinoma in high-risk area of Thailand: role of lifestyle, diet and methylenetetrahydrofolate reductase polymorphisms

Background and aim: Cholangiocarcinoma (CCA) is the most common cancer in Northeast Thailand. Endemicity of Opisthorchis viverrini (OV) – a known carcinogen – is responsible, but although infection is very common, the lifetime risk of CCA is only 5%. Other co-factors must exist, including aspects of lifestyle or diet along with variations in genetic susceptibility to them. Change in methylenetetrahydrofolate reductase (MTHFR) activity may influence both DNA methylation and synthesis. This study investigates risk factors for CCA with a focus on lifestyle, diet and MTHFR polymorphisms. Methods: Nested case–control study within cohort study was conducted. 219 subjects with primary CCA were each matched with two non-cancer controls from the same cohort on sex, age at recruitment and presence/absence of OV eggs in stool. Lifestyle and dietary data were obtained at recruitment. MTHFR polymorphisms were analyzed using PCR with high resolution melting analysis. The associations were assessed using conditional logistic regression. Results: Consumption of alcohol, raw freshwater fish and beef sausage increased the risk of CCA, while fruit and/or vegetables consumption reduced risk. There were interactions between MTHFR and preserved freshwater fish and beef. These dietary items are either a source of OV or of pre-formed nitrosamine, folate and antioxidants that are of possible relevance in OV carcinogenesis. Conclusions: Primary prevention of CCA in high-risk population is based upon efforts to reduce OV infection. Reduced consumption of alcohol and preserved meats, and increased consumption of dietary folate, actions with a wider preventive potential, may also help in the reduction of CCA burden.     

A rational relationship: Oncolytic virus vaccines as functional partners for adoptive T cell therapy

Tumours employ a variety of immune-evasion and suppression mechanisms to impair development of functional tumor-specific T cells and subvert T cell-mediated immunity in the tumour microenvironment. Adoptive T cell therapy (ACT) aims to overcome these barriers and overwhelm tumor defenses with a bolus of T cells that were selectively expanded ex vivo. Although this strategy has been effective in liquid tumors and melanomas, many tumors appear to be resistant to ACT. Several factors are thought to play into this resistance, including poor engraftment and persistence of transferred cells, tumour cell heterogeneity and antigen loss, poor immune cell recruitment and infiltration into the tumour, and susceptibility to local immunosuppression in the tumor microenvironment. Oncolytic viruses (OV) have been identified as powerful stimulators of the anti-tumour immune response. As such, OVs are inherently well-positioned to act in synergy with ACT to bolster the anti-tumour T cell response. Further, OV vaccines, wherein tumour-associated antigens are encoded into the viral backbone, have proven to be remarkable in boosting antigen-specific T cell response. Pre-clinical studies have revealed remarkable therapeutic outcomes when OV vaccines are paired with ACT. In this scenario, OV vaccines are thought to function in a “push and pull” manner, where push refers to expanding T cells in the periphery and pull refers to recruiting those cells into the tumour that has been rendered amenable to T cell attack by the actions of the OV. In this review, we discuss barriers that limit eradication of tumors by T cells, highlight attributes of OVs that break down these barriers and present strategies for rational combinations of ACT with OV vaccines.