Thymidine Kinase Research Articles

Epidemiological survey of bovine gammaherpesvirus 4 (BoHV-4) infection in cattle and buffalo from Pakistan.

This study was conducted to evaluate the prevalence of bovine gammaherpesvirus 4 (BoHV-4) among healthy cattle and buffaloes as well as those associated with different diseases (respiratory tract infection, mastitis and reproductive tract infection) in District Chakwal, Pakistan. Blood, swab and milk samples of cattle and buffaloes were randomly collected from different areas of Chakwal. DNA was isolated from the samples and subjected to nested PCR using thymidine kinase gene primers. Out of 300 samples (200 blood, 50 swab and 50 milk samples) from both species (cattle and buffalo), an overall prevalence of BoHV-4 of 3.33% was obtained. Samples from cattle showed a higher species-specific prevalence (4.16%) than samples from buffalo (2.78%). One sample out of 50 swab samples and 1 out of 50 milk samples were also positive for BoHV-4. DNA sequencing of a positive PCR product from cattle confirmed that the sequence was from the thymidine kinase gene of BoHV-4. Phylogenetic analysis also revealed close similarities with other BOHV-4 thymidine kinase sequences. To detect BoHV-4 antibodies, an indirect ELISA was also performed. Two hundred blood samples were also collected from the same animals in nonanticoagulant-containing tubes for the isolation of serum and were subjected to indirect ELISA. Sixteen samples (8%) were positive for BoHV-4 antibodies. This study will be useful in further diagnoses of BoHV-4 in Pakistan and in devising measures to control the spread of BoHV-4.

Nonclinical safety assessments of a novel synthetic toll-like receptor 4 agonist and saponin based adjuvant

A full nonclinical safety package was performed to support the clinical use of SPA14, a novel liposome-based vaccine adjuvant containing the synthetic toll-like receptor 4 agonist E6020 and saponin QS21.E6020 and QS21 were tested negative for their potential genotoxic effects in Ames, micronucleus, or mouse-lymphoma TK (thymidine kinase) assay. To evaluate the potential local and systemic effects of SPA14, two toxicity studies were performed in rabbits. In the first dose range finding toxicity study, rabbits received two intramuscular injections of SPA14 at increasing doses of E6020 combined with two antigens, a control (saline), the two antigens alone, or the antigens adjuvanted with a liposome-based adjuvant AS01B. No systemic toxicity was detected, supporting the dose of 5 μg of E6020 for the subsequent pivotal study. In the second repeated dose toxicity study, rabbits received four intramuscular injections of SPA14 alone, a control (saline), SPA14 combined with two antigens, the two antigens alone, or the antigens combined with AF03 adjuvant, which is a squalene-based emulsion. SPA14 alone or in combination with the antigens was well tolerated and did not cause any systemic toxicity. Finally, two safety pharmacology studies were conducted to assess potential cardiovascular and respiratory effects of E6020 and SPA14 in conscious telemetered cynomolgus monkeys and beagle dogs, respectively. One subcutaneous injection of E6020 in monkeys and one intramuscular injection of SPA14 in dogs had no consequences on respiratory and cardiovascular functions. Altogether these results support the clinical development of SPA14.

P16-EXPRESSING SENESCENT CELLS ARE A DOUBLE-EDGED SWORD IN SHAPING IMMUNE RESPONSES WITH AGE

Abstract Aging results in the accumulation of senescent cells which can cause dysfunction in many contexts but the effects on immune responses remain unclear. Here, we aimed to probe the effects of clearing senescent cells in aged mice on the immune response to influenza infection. We utilized a powerful p16 trimodality reporter mouse model (p16-3MR): under the control of the p16 promoter, these mice express cassettes encoding luciferase, RFP, and herpesvirus thymidine kinase (HSV-TK). p16 is commonly upregulated in senescent cells so this model allows us to selectively delete those cells by treating with ganciclovir (GCV), which will induce apoptosis in cells expressing HSV-TK. We hypothesized that while p16-expressing senescent cells may exacerbate dysfunctional responses to a primary infection, they may play a protective role in resolving inflammation and fostering memory cell generation. We found that deletion of p16-expressing cells enhanced viral clearance and decreased infiltration of pro-inflammatory flu-specific CD8 T cells during the primary response to infection. Conversely, at 30 days post infection, there were fewer flu-specific CD8 memory T cells and lower amounts of anti-viral antibodies in the lungs of GCV treated mice. We also observed perturbations in memory T cell trafficking in GCV treated mice. Furthermore, GCV treated mice were unable to mount an effective memory response and were unable to control viral load following a heterosubtypic challenge. This suggests that targeting senescent cells may potentiate primary responses while limiting the ability to form durable and protective immune memory with age.

Development of Allogeneic Stem Cell-Based Platform for Delivery and Potentiation of Oncolytic Virotherapy.

We describe the repurposing and optimization of the TK-positive (thymidine kinase) vaccinia virus strain ACAM1000/ACAM2000™ as an oncolytic virus. This virus strain has been widely used as a smallpox vaccine and was also used safely in our recent clinical trial in patients with advanced solid tumors and Acute Myeloid Leukemia (AML). The vaccinia virus was amplified in CV1 cells and named CAL1. CAL1 induced remarkable oncolysis in various human and mouse cancer cells and preferentially amplified in cancer cells, supporting the use of this strain as an oncolytic virus. However, the therapeutic potential of CAL1, as demonstrated with other oncolytic viruses, is severely restricted by the patients' immune system. Thus, to develop a clinically relevant oncolytic virotherapy agent, we generated a new off-the-shelf therapeutic called Supernova1 (SNV1) by loading CAL1 virus into allogeneic adipose-derived mesenchymal stem cells (AD-MSC). Culturing the CAL1-infected stem cells allows the expression of virally encoded proteins and viral amplification prior to cryopreservation. We found that the CAL1 virus loaded into AD-MSC was resistant to humoral inactivation. Importantly, the virus-loaded stem cells (SNV1) released larger number of infectious viral particles and virally encoded proteins, leading to augmented therapeutic efficacy in vitro and in animal tumor models.

Markers for internal neoplasia in the horse

The diagnosis of internal neoplasia in horses is challenging. Increased production of hormones physiologic for adult animals (e.g., adrenocorticotropin, norepinephrine, and erythropoietin) or typical for the foetal phase (alpha-fetoprotein, anti-Müllerian hormone, and parathyroid-hormone-related protein) might aid in tumour diagnostics. Thymidine kinase-1 and alkaline phosphatase are examples of intracellular enzymes, whose activity in the blood may increase in some neoplasia cases. Furthermore, inappropriate production of abnormal monoclonal or autologous antibodies can accompany lymphoma and multiple myeloma. Many of those tumour markers lead to clinical or laboratory changes, called paraneoplastic syndromes, such as hypercalcaemia and erythrocytosis. The interpretation of the results of the tumour marker measurements in horses is complicated due to many factors affecting the markers' concentration or activity (e.g., young age, pregnancy, and inflammation) and other diseases triggering the same changes. Moreover, the presence of paraneoplastic syndromes is inconsistent, which leads to low sensitivity of those substances as tumour markers. In conclusion, screening for neoplasia in horses is not recommended. The measurement of tumour markers should be performed only in risk groups with suspicious clinical or laboratory findings, and the results should be interpreted with caution. It is advisable to add inflammatory markers to the tumour profile or repeat the measurements.

Stimulus-responsive strategy based on MnO2 nanosheet-modified mesoporous silica nanoprobes for accurate multiple mRNAs detection

Fluorescence detection of multiple mRNAs has attracted great attention for disease diagnosis. In this work, a stimulus-responsive strategy for highly sensitive and accurate multiple mRNAs detection was proposed. This stimulus-responsive detection system was prepared by mesoporous silica nanoparticles (MSN), manganese dioxide (MnO2) nanosheets, and DNA probes. DNA probes were loaded into the pores of MSN, which were closed with MnO2 nanosheets. In the presence of glutathione (GSH) and target mRNAs, MnO2 nanosheets were degraded by GSH, resulting in the release of DNA probes. These DNA probes hybridized to the corresponding target mRNA, thereby changing the fluorescence intensity of fluorophores of DNA probes, which could achieve the quantification of target mRNA. This system could simultaneously detect survivin mRNA and Thymidine kinase 1 mRNA at low background levels with relative limits of detection of 0.9 nM and 0.7 nM, respectively. Moreover, this assay has been successfully applied to detect multiple mRNAs with adequate anti-interference ability in the biological sample.

In silico investigation of acyclovir derivatives potency against herpes simplex virus

Herpes simplex virus (HSV) is a major human pathogen, sub-divided into two types: HSV-1 which causes oral and perioral infections and HSV-2 which causes genital herpes. The global disease burden is high with 67% and 11% of the World's population being infected with HSV-1 and HSV-2 respectively. Acyclovir, an FDA approved drug, is a synthetic purine nucleoside analogue used for the treatment of HSV infections. The drug acts via the viral encoded thymidine kinase and competitively inhibits viral DNA polymerase. Emerging HSV resistance to acyclovir and lack of approved vaccines necessitates need for effective strategies to circumvent the viral infection. Derivatives of acyclovir were modelled using computer aided drug design techniques through Chemsketch and drugability determined based on Lipinski's Ro5 where a Python filtering code was created and derivatives with zero non-violations were selected. Putative biological targets were determined through network pharmacology and validated through gene ontology. Molecular docking with known and putative targets was done to determine the binding affinities of the acyclovir derivatives. Based on enzyme inhibition scores, pharmacokinetics prediction was done on Molinspiration while pharmacodynamics predictions were done on AdmetSAR based on bioavailability scores. Thirty acyclovir derivatives were modelled of which 22 had zero non-violations. 2-[(3, 6-dihydro-9H-purin-9-yl)methoxy]ethan-1-ol) had the highest enzyme inhibition score at 1.0 compared to 0.84 for acyclovir. The molecule was further optimized generating (2‐[(6‐methyl‐6,9‐dihydro‐3H‐purin‐9‐yl)methoxy]ethan‐1‐ol which had a better bioavailability and a higher blood brain barrier value of 0.9880 indicating possible better patient tolerance. 2‐[(6‐methyl‐6,9‐dihydro‐3H‐purin‐9‐yl)methoxy]ethan‐1‐ol had an enzyme inhibition score of 0.90 and binding energies of -5.1 kcal/mol when docked against thymidine kinase. The intra-viral HSV PPI network was analysed and DNA replication helicase (UL5) and serine /threonine-protein kinase (US3) were selected due to functional similarity with thymidine kinase with kappa statistics value of 0.30 and 0.24 respectively. Gene enrichment analysis indicated that the biological targets UL5 and US3 had a significant P value of 0.62 based on Benjamin Correction at P<0.05. The study recommends in vitro and in vivo validation of this novel compound and molecular validation of the two new potential biological targets for possible development of new anti-herpes therapeutic compounds.

A small molecule binding to TGGAA pentanucleotide repeats that cause spinocerebellar ataxia type 31

Spinocerebellar ataxia type 31 is an autosomal dominant neurodegenerative disease caused by aberrant insertion of d(TGGAA)n into the intron shared by brain expressed, associated with Nedd4 and thymidine kinase 2 genes in chromosome 16. We reported that a naphthyridine dimer derivative with amidated linker structure (ND-amide) bound to GGA/GGA motifs in hairpin structures of d(TGGAA)n. The binding of naphthyridine dimer derivatives to the GGA/GGA motif was sensitive to the linker structures. The amidation of the linker in naphthyridine dimer improved the binding property to the GGA/GGA motif as compared with non-amidated naphthyridine dimer.

Treatment with hepatocyte transplantation in a novel mouse model of persistent liver failure

Background and AimCell-based transplantation therapy using hepatocytes, hepatic stem cells, hepatocyte-like cells induced from stem cells, etc. is thought to be an attractive alternative to liver transplantation, and have been studied to date. For its clinical application, however, it is extremely important to develop a model that reproduces the pathological conditions with indication for treatment and enables the study for the ideal treatment strategy. MethodsThe transgenic mice which express the thymidine kinase (TK) gene of human herpes simplex virus (HSV) in their hepatocytes with normal immunity has been developed (designated as HSVtk). After ganciclovir (GCV) administration which injure TK-expressing hepatocytes, the primary hepatocytes (PHs) isolated from green fluorescent protein (GFP) transgenic mouse (GFP-tg) were transplanted to HSVtk intrasplenically, and replacement index (RI) with transplanted PHs in the liver, liver histology, and mRNA expressions in the liver were analyzed up to 8 weeks after transplantation. ResultsHSVtk without PH transplantation after GCV administration developed persistent liver failure with degenerated hepatocytes, persistent elevation of ALT and hepatic p16 mRNA levels, suggesting the existence of cellular senescence in the base of the disease. When autologous GFP-PHs were transplanted to HSVtk, the transplanted cells were successfully engrafted in the liver. Eight weeks after transplantation, serum ALT levels and liver histology were almost normalized, while RIs varied from 19.8 to 73.8%. Since the hepatic p16 mRNA levels were decreased significantly in these mice, the senescence of hepatocytes associated with liver injury was thought to be resolved. On the other hand, allogenic GFP-PHs transplanted to HSVtk were eliminated as early as 1 week after transplantation. In these mice, hepatic p16 mRNA levels were significantly increased at 8 weeks after transplantation, suggesting the aggravation of hepatocyte senescence. FK506 administration to HSVtk protected the transplanted hepatocytes with allogenic background from rejection at 2 weeks after transplantation, but the condition of mice and the senescent status in the liver seemed worsened. ConclusionsThe mouse model with HSVtk/GCV system was useful for studying the mechanism of liver regeneration and the immune rejection responses in the hepatocyte transplantation treatment. It may also be utilized to develop the effective remedies to avoid immune rejection.

Fludarabine, Cyclophosphamide and Rituximab (FCR) As First Line Treatment in Patients with Chronic Lymphocytic Leukemia (CLL): A Long-Term Analysis of the German CLL Study Group (GCLLSG) Registry

Introduction: Large randomized trials investigating targeted agents have resulted in a markedly improved progression-free survival (PFS) when compared to standard chemoimmunotherapy. However, for small subgroups with a favorable risk profile, overall survival was not consistently superior for targeted agents. Long-term follow up on FCR suggested that favorable risk patients (pts) benefit significantly from this therapy and can expect an exceptionally long PFS. In addition, targeted first line (1L) therapies for favorable risk pts are reimbursed only in a minority of countries. Here, we report long-term efficacy data of FCR treatment obtained from pts documented within the GCLLSG registry. Methods: Documentation within the GCLLSG registry started in 2013 and included both pts from prospective trials (29.7%), most importantly of the CLL8 trial (22.6%) and CLL10 trial (62.1%) as well as retrospective data. Pts with confirmed diagnosis of CLL who received FCR as 1L treatment were included. Patient characteristics were reported in median, interquartile ranges or frequencies and percentages. Response was reported as response or non-response. Event-free survival (EFS), which was defined as the time from start of (first) FCR treatment to disease progression, initiation of subsequent treatment for CLL or death. EFS was chosen as an endpoint for this report due to concerns that PFS might give unrealistically good results due to lack of documentation. However, PFS and TTNT data will be presented at the meeting. Overall survival was calculated from initiation of 1L treatment for all pts who received 1L treatment after 2013. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated by Cox proportional hazards regression. Variables that were significantly associated with EFS in univariate analyses (significance level was set at 5%) were considered as candidates for the multivariate modelling to identify independent prognostic risk factors. Results: As of September 2021, a total of 5922 pts were enrolled in the registry of which 417 received FCR as 1L treatment between 2003 and 2021. 223 pts (53.5%) received treatment before 2013 and 194 (46.5%) after 2013. Median observation time from 1L treatment was 95.8 (interquartile range 58.7-126.8) months. Patient characteristics showed a median age of 58 (range 24-83) years. Unmutated IGHV (U-CLL) was present in 61.4% of the pts. Regarding genomic aberrations based on the hierarchical model, del(17p) was found in 1.9%, del(11q) in 15.2%, trisomy12 in 15.5%, mono-allelic del(13q) in 34.3% and none of these changes in 33.0% of the pts. 88.8% of the pts responded to first line therapy, 11.2% were documented as non-responder. Median EFS was 71.1 month, for 188 pts no event was documented at the time point of data cut-off. Age group (≤ 65 years, >65 years), del(11q), IGHV mutational status, TP53 status, and serum thymidine kinase (TK) were associated with EFS in univariate analyses. Overall, 116 pts with 67 events were included in the final multivariable model for EFS. U-CLL (HR 1.949 (95%CI: 1.170-3.247; p=0.010) and TK >10 U/l (HR 2.107 (95%CI: 1.153-3.849; p=0.015) were independent adverse prognostic risk factors for EFS. When low TK and M-CLL were combined, the median EFS was not reached, with a 5-year survival rate of 95.8%. In contrast, median EFS was 84.1 months for pts with U-CLL and high TK. In U-CLL pts, median EFS was 87.0 months for pts with TK ≤ 10 U/l, and 58.9 months for pts with TK > 10 U/l. Median overall survival was not reached with a 5-year survival rate of 92.7%. Conclusion: Within this cohort of pts with a long follow up in the GCLLSG registry, FCR treatment was associated with a long EFS especially in pts with a low risk profile exhibiting a mutated IGHV status und low TK. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Manipulation of Thymidine Nucleotide Metabolism Controls Human Telomere Length and Promotes Telomere Elongation in Dyskeratosis Congenita Patient Derived Cells

Inherited mutations in telomere maintenance genes are associated with short telomeres and can cause severe diseases including the congenital bone marrow failure syndrome dyskeratosis congenita, liver cirrhosis, and pulmonary fibrosis, collectively referred to as telomere biology disorders (TBDs). Current approaches to treat TBDs rely on bone marrow and organ transplantation, and patients face poor prognoses. New systemic therapies to promote telomere elongation and treat TBDs throughout the body are needed. Here, we performed the first genome-wide CRISPR/Cas9 functional telomere length screen using flow cytometry-FISH (flow-FISH) in human cells to identify novel telomere maintenance pathways that might be manipulated for therapeutic benefit. Validating our approach, we found guide RNAs targeting positive (e.g. TERT) and negative (e.g. POT1 and TRF1) regulators of telomere length enriched in sorted cells with short and long telomeres, respectively. Unbiased pathway analysis of screening hits demonstrated nucleotide metabolism genes to be highly associated with telomere length, consistent with recent genome-wide association studies, and which in follow-up screening we narrowed down to multiple control points specifically along the thymidine (dT) nucleotide metabolism pathway. Targeted genetic disruption using CRISPR/Cas9 revealed that decreasing dT nucleotide salvage by thymidine kinase (TK1) or de novo production by thymidylate synthase (TYMS) reduced telomere length, whereas inactivation of the dNTPase-encoding gene SAMHD1 lengthened telomeres. We next asked whether nucleotide precursor manipulation could alter telomere length dynamics. Remarkably, we found supplementation with dT nucleoside alone drove rapid telomere elongation in human cells (Figure 1). Telomere elongation after dT treatment required conversion to thymidine monophosphate (dTMP) by TK1, was telomerase dependent, and could be achieved at doses that did not inhibit cell cycle progression. In cells with TYMS deletion or after treatment with the TYMS inhibitor 5-fluorouracil, dT supplementation was sufficient to bypass the de novo nucleotide synthesis defect and promoted robust telomere elongation. SAMHD1 overexpression blunted the degree of telomere elongation from dT treatment in a manner requiring its dNTPase activity, suggesting that telomere length control occurs at the level of dTTP in human cells. To assess whether nucleotide manipulation could overcome the genetic causes of telomere insufficiency which drive TBDs, we tested dT on induced pluripotent stem cell (iPSC) lines derived from patients harboring mutations in a spectrum of TBD-associated genes including DKC1, TERC, TERT, TINF2, RTEL1, and PARN. We found dT supplementation or inhibition of SAMHD1 drove robust telomere elongation in patient derived iPSCs spanning genotypes that cause telomere maintenance defects both by telomerase-dependent and telomerase-independent mechanisms. In preliminary experiments using primary human hematopoietic stem and progenitor cells, dT treatment resulted in telomere elongation in vitro, pointing to a potential treatment for TBD-associated bone marrow failure. Collectively, our results demonstrate a critical role of thymidine nucleotide metabolism in regulating human telomere length. Based on our findings, we propose there may be a therapeutic window to modulate telomere length by manipulating dT metabolism in patients with TBDs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Tumor and stroma COL8A1 secretion induces autocrine and paracrine progression signaling in pancreatic ductal adenocarcinoma

Several collagen subtypes are involved in pancreatic ductal adenocarcinoma (PDAC) desmoplasia, which constrains therapeutic efficacy. We evaluated collagen type VIII alpha 1 chain (COL8A1), whose function in PDAC is currently unknown. We identified COL8A1 expression in 7 examined PDAC cell lines by microarray analysis, western blotting, and RT‒qPCR. Higher COL8A1 expression occurred in 2 gemcitabine-resistant PDAC cell lines; pancreas tissue (n=15) from LSL-KrasG12D/+; p48-Cre mice with advanced PDAC predisposition; and PDAC parenchyma and stroma of a patient tissue microarray (n=82). Bioinformatic analysis confirmed higher COL8A1 expression in PDAC patient tissue available from TCGA (n=183), GTEx (n=167), and GEO (n=261) databases. siRNA or lentiviral sh-mediated COL8A1 inhibition in PDAC cells reduced migration, invasion and gemcitabine resistance and resulted in lower cytidine deaminase and thymidine kinase 2 expression and was rescued by COL8A1-secreting cancer-associated fibroblasts (CAFs). The activation of COL8A1 expression involved cJun/AP-1, as demonstrated by CHIP assay and siRNA inhibition. Downstream of COL8A1, activation of ITGB1 and DDR1 receptors and PI3K/AKT and NF-κB signaling occurred, as detected by expression, adhesion and EMSA binding studies. Orthotopic transplantation of PDAC cells with downregulated COL8A1 expression resulted in reduced tumor xenograft growth and lower gemcitabine resistance but was prevented by cotransplantation of COL8A1-secreting CAFs. Most importantly, COL8A1 expression in PDAC patient tissues from our clinic (n=84) correlated with clinicopathological data, and we confirmed these findings by the use of patient data (n=177) from the TCGA database. These findings highlight COL8A1 expression in tumor and stromal cells as a new biomarker for PDAC progression.

Proteome-Based Investigation Identified Potential Drug Repurposable Small Molecules Against Monkeypox Disease.

Monkeypox Virus (MPXV), the causative agent of Monkeypox (MPX) disease, is an emerging zoonotic pathogen spreading in different endemic and non-endemic nations and creating outbreaks. MPX treatment mainly includes Cidofovir and Tecovirimat but they have several side effects and solely depending on these drugs may promote the emergence of drug-resistant variants. Hence, new drugs are required to control the spread of the disease. In this study, we explored the MPXV proteome to suggest repurposable drugs. DrugBank screening revealed drugs such as Brinzolamide, Dorzolamide, Methazolamide, Zidovudine, Gemcitabine, Hydroxyurea, Fludarabine, and Tecovirimat as controls. Structural analogs of these compounds were extracted from ChEMBL Database. After Molecular docking and Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET)-based screening, we identified Zidovudine (binding affinity-5.9kcal/mol) and a Harmala alkaloid (2S,4R)-4-(9H-Pyrido[3,4-b]indol-1-yl)-1,2,4-butanetriol (binding affinity -6.6kcal/mol) against L2R receptor (Thymidine Kinase). Moreover, Fludarabine (binding affinity -6.4kcal/mol) and 5'-Dehydroadenosine (binding affinity -6.4kcal/mol) can strongly interact with the I4L receptor (Ribonucleotide reductase large subunit R1). Molecular Dynamics (MD) simulations suggest all of these compounds can change the C-alpha backbone, residue mobility, compactness, and solvent accessible surface area of L2R and I4L. Our results strongly suggest that these drug repurposing small molecules are worth exploring in vivo and in vitro for clinical applications.

Intracellular Multicomponent Synchronous DNA-Walking Strategy for the Simultaneous Quantification of Tumor-Associated Proteins in a Single Cell.

Single-cell protein analysis is very important for understanding cellular heterogeneity and single-cell biology. However, owing to the extremely low levels of some tumor-associated proteins in individual cells, the absolute quantification of such tumor-associated proteins in a single cell remains a challenge. Herein, an intracellular multicomponent synchronous DNA-walking strategy is proposed for the simultaneous quantification of multiple tumor-associated proteins in a single cell. In this strategy, a nanoprobe based on a DNA walker was designed for the simultaneous signal amplification of nucleolin (NCL) and thymidine kinase 1 (TK1) in a single cell. NCL and TK1 in single cells were simultaneously detected on a microchip platform with detection limits of 1.0 and 0.8 pM, respectively. The results obtained from 20 liver cancer cells (HepG2) and 20 normal hepatocytes (HL-7702) indicated that NCL and TK1 were overexpressed in liver cancer cells. However, the levels of NCL and TK1 in normal hepatocytes are only about one-tenth to one-sixth of those in hepatic carcinoma. Using different nucleic acid aptamers, the proposed strategy can be applied for the analysis of other single-cell proteins and in the early diagnosis of cancer.

Antiviral Potentialities of Chemical Characterized Essential Oils of Acacia nilotica Bark and Fruits against Hepatitis A and Herpes Simplex Viruses: In Vitro, In Silico, and Molecular Dynamics Studies.

Acacia nilotica (synonym: Vachellia nilotica (L.) P.J.H.Hurter and Mabb.) is considered an important plant of the family Fabaceae that is used in traditional medicine in many countries all over the world. In this work, the antiviral potentialities of the chemically characterized essential oils (EOs) obtained from the bark and fruits of A. nilotica were assessed in vitro against HAV, HSV1, and HSV2. Additionally, the in silico evaluation of the main compounds in both EOs was carried out against the two proteins, 3C protease of HAV and thymidine kinase (TK) of HSV. The chemical profiling of the bark EOs revealed the identification of 32 compounds with an abundance of di- (54.60%) and sesquiterpenes (39.81%). Stachene (48.34%), caryophyllene oxide (19.11%), and spathulenol (4.74%) represented the main identified constituents of bark EO. However, 26 components from fruit EO were assigned, with the majority of mono- (63.32%) and sesquiterpenes (34.91%), where trans-caryophyllene (36.95%), Z-anethole (22.87%), and γ-terpinene (7.35%) represented the majors. The maximum non-toxic concentration (MNTC) of the bark and fruits EOs was found at 500 and 1000 µg/mL, respectively. Using the MTT assay, the bark EO exhibited moderate antiviral activity with effects of 47.26% and 35.98% and a selectivity index (SI) of 2.3 and 1.6 against HAV and HSV1, respectively. However, weak activity was observed via the fruits EO with respective SI values of 3.8, 5.7, and 1.6 against HAV, HSV1, and HSV2. The in silico results exhibited that caryophyllene oxide and spathulenol (the main bark EO constituents) showed the best affinities (ΔG = -5.62, -5.33, -6.90, and -6.76 kcal/mol) for 3C protease and TK, respectively. While caryophyllene (the major fruit EO component) revealed promising binding capabilities against both proteins (ΔG = -5.31, -6.58 kcal/mol, respectively). The molecular dynamics simulation results revealed that caryophyllene oxide has the most positive van der Waals energy interaction with 3C protease and TK with significant binding free energies. Although these findings supported the antiviral potentialities of the EOs, especially bark EO, the in vivo assessment should be tested in the intraoral examination for these EOs and/or their main constituents.

Preclinical gene therapy in glioblastoma multiforme: Using olfactory ensheathing cells containing a suicide gene

AimsGlioblastoma multiforme (GBM) is the most malignant type of brain tumor resistant to current treatments. Recently, suicide gene therapy with the Herpex Simplex Virus thymidine kinase (HSV-tk) gene has been developed with high therapeutic potency, even in clinical trials. The primary challenge to establishing a gene therapy strategy is how to transfer the desired gene into the tumor site. The olfactory ensheathing cells (OECs) secreting neurotropic and anti-inflammatory factors have a high migration capacity, making them applicable for gene therapy. We examined our new construct OECs containing the HSV-tk gene for their migration and tumoricidal ability in animal models of GBM. Main methodsIsolated OECs were transduced by the HSV-tk gene (OEC-tks). OEC-tks or PBS were injected ipsilaterally or contralaterally into the tumor-bearing rats, followed by gancyclovir (GCV) or PBS administration. At the end of the treatment, tumor size, apoptosis, and animal survival were assessed. Key findingsOur findings demonstrated that tumor size was significantly decreased in OEC-tks ipsilateral and contralateral groups, followed by GCV injections. Furthermore, both groups' pro-apoptotic protein and gene expressions were up-regulated, whereas Bcl-2 protein expression was down-regulated. Besides, apoptosis in the OEC-tks ipsilateral/GCV group was higher in the intratumoral region, and this percentage was higher in the OEC-tks contralateral/GCV group in the peritumoral region. Interestingly, our new construct increased animal survival rate and reduced body weight loss. SignificanceOECs could serve as a novel carrier for gene therapy, have a high migration capability to the GBM and eventually suppress tumor progression.

Detection and molecular characterization of infectious laryngotracheitis virus (ILTV) in chicken with respiratory signs in Brazil during 2015 and 2016.

Avian infectious laryngotracheitis (ILT) is a respiratory disease that causes severe economic losses in the poultry industry, mainly due to high morbidity and mortality and reduced egg production. Molecular characterization was performed on samples collected from flocks in the Brazilian States of São Paulo, Pernambuco, and Minas Gerais during 2015 and 2016 that presented clinical signs of respiratory disease. End-point PCR was used for viral detection, and DNA sequencing was used for differentiation of vaccine and field strains. Molecular analysis based on the infected cell protein (ICP4) gene separated four of the nine samples together with previous Brazilian isolates (São Paulo and Minas Gerais), one sample was grouped on the same branch as Minas Gerais strains (along with another related sample), one sample was separately branched but still related to the tissue culture origin (TCO) vaccine strain, and two samples were grouped on the same branch as the TCO vaccine strain. Molecular analysis of the thymidine kinase (TK) gene showed the existence of strains of both high and low virulence. The characterization of two fragments of the ICP4 gene and a fragment of the TK gene in this study suggested that the virus circulating in Guatapará, as well as those in Barretos and Itanhandu, that is causing respiratory problems in birds is a highly virulent field strain. The clinical signs point to a TCO vaccine strain that most likely underwent some reversal event and is a latent reactivated infection.

GC/MS profiling of essential oils from Bontia daphnoides L., chemometric discrimination, isolation of dehydroepingaione and evaluation of antiviral activity

Bontia daphnoides L. has been utilized in traditional medicine for treatment of herpes, cough and colds. The aim of this study was to analyze the volatile constituents of this plant by GC/MS (Gas Chromatography coupled to Mass Spectrometry) and to assess their antiviral activity. A total of 64 compounds were identified where dehydroepingaione represented 83.60, 72.36, 58.78 and 34.18% in the leaves, stems, flowers and fruits, respectively. Principal component and hierarchical cluster analysis revealed the discrimination of the organs as the leaves and stems were distributed in the same cluster in contrast to the flowers and fruits. Furthermore, the antiviral activity was assessed where the oils of leaves and stems exhibited potent antiviral activity displaying IC50 of 11.98, 12.62 µg/ml against HSV-1 and 13.34, 14.50 µg/ml against CoxB4, respectively. Dehydroepingaione was isolated from the n-hexane fraction of the leaves and showed activity against HSV-1 and CoxB with IC50 of 24.46 and 25.32 µg/ml, respectively. Molecular modelling studies illustrated that the major compounds showed good affinity towards HSV type-1 thymidine kinase. Therefore, it can be concluded that the oils from B. daphnoides have promising antiviral activity that may be attributed to the major oxygenated sesquiterpenes.

DNA-damaging cancer cells to improve virotherapy

In the current issue of MTO, an interesting work by Koch and colleagues reveals that perturbing the DNA damage response (DDR) signaling pathway enhances the cytotoxic effect of local treatment with CAN-2409, a first-generation adenoviral vector encoding the suicide gene herpes simplex virus thymidine kinase (HSV-tk), which in the presence of ganciclovir causes DNA damage, and immunogenic cell death and that is already in clinical trials in high-grade gliomas.1,2,3 The authors evaluate the efficacy of ATR/ATM inhibitors in combination with the virus in vitro and in vivo in immunocompetent mouse models to decipher its effect in tumor immune microenvironment using state-of-the-art technologies such as cytometry by time of flight (CyTOF).

External Validation of the Colon Life Nomogram for Predicting 12-Week Mortality in Dutch Metastatic Colorectal Cancer Patients Treated with Trifluridine/Tipiracil in Daily Practice.

Simple SummaryPredicting prognosis in cancer patients is needed to guide decision making. In order to predict survival, nomograms can be used to estimate chances of survival based on clinical characteristics. In order to identify metastatic colorectal cancer (mCRC) patients with a very short life expectancy (less than 12 weeks) after receiving multiple standard treatments, the Colon Life nomogram was previously developed. Before a nomogram can be used in daily practice, it is essential to show that it accurately predicts survival in different real-life populations and can be used to guide clinical decision making. This is called external validation. We externally validated the Colon Life nomogram in a cohort of patients with refractory mCRC who were treated with a last treatment option, trifluridine/tipiracil, in daily practice. We demonstrated that the nomogram severely overestimated 12-week mortality and therefore should not be used in clinical practice in its present form. We also showed that quality of life reported by patients themselves can improve the prediction of survival, stressing the importance of patient-reported outcomes. We recommend conducting a study with a sufficiently large sample size to update the Colon Life nomogram or to develop a new model and include quality of life.Background: Predicting prognosis in refractory metastatic colorectal cancer (mCRC) patients is needed to guide decision making. The Colon Life nomogram was developed to predict 12-week mortality in refractory mCRC patients. The aim of this study is to validate the Colon Life nomogram in last line/refractory patients receiving trifluridine/tipiracil (FTD/TPI) in daily practice. Methods: The validation cohort consists of 150 QUALITAS study patients, an observational substudy of the Prospective Dutch CRC cohort, who were treated with FTD/TPI between 2016 and 2019. Model performance was assessed on discrimination, calibration, and clinical usefulness. The additional prognostic value of baseline quality of life (QoL) and thymidine kinase (TK1) expression in tissue was explored. Results: Of the 150 patients, 25 (16.7%) died within 12 weeks of starting FTD/TPI treatment. The C-statistic was 0.63 (95% C.I. 0.56–0.70). The observed/expected ratio was 0.52 (0.37–0.73). The calibration intercept and slope were −1.06 (−1.53 to −0.58) and 0.41 (0.01–0.81), respectively, which indicated overestimation of 12-week mortality by the nomogram. Decision curve analysis showed the nomogram did not yield a positive net benefit at clinically meaningful thresholds for predicted 12-week mortality. Addition of QoL to the nomogram improved the C-statistic to 0.85 (0.81–0.89). TK1 expression was associated with progression-free survival but not with overall survival. Conclusion: We demonstrated evident miscalibration of the Colon Life nomogram upon external validation, which hampers its use in clinical practice. We recommend conducting a study with a sufficiently large sample size to update the Colon Life nomogram or to develop a new model including QoL.