scholarly journals The characterisation and application of bacterial nitroreductase enzymes

2021 ◽  
Author(s):  
◽  
Sarah Condon
Keyword(s):  
New Technologies ◽  
Active Form ◽  
Model Organism ◽  
Eukaryotic Cell ◽  
Screening Process ◽  
Enzyme Prodrug Therapy ◽  
Transgenic Model ◽  
Cell Ablation ◽  
Anti Cancer ◽  
Safety Features

<p>Cancer is an increasing global concern, with the number of people diagnosed growing rapidly each year. Gene directed enzyme prodrug therapy (GDEPT) is emerging as a front-runner of new technologies that seek to combat the growing number of cases. One developing approach to GDEPT involves the use of bacterial nitroreductase enzymes to reduce prodrug substrates, which, upon reduction to their active form, are toxic to cancer cells through DNA crosslinking.  Nitroreductases have the ability to activate a variety of nitro-quenched compounds, not only anti-cancer prodrugs, but also nil bystander antibiotics and masked fluorophores, through the reduction of strongly electron-withdrawing nitro substituents on aromatic rings. My research initially sought to exploit this capability by partnering nitroreductases with nil bystander antibiotics for targeted cell ablation, as a component of a larger gene directed enzyme prodrug therapy project. This has potential to provide important safety features for removal of viral and bacterial vectors following anti-cancer gene therapy.  From this, the main focus evolved into utilising nitroreductase enzymes for targeted cell ablation for applications in developmental and regenerative biology. This exploited the ability of nitroreductases to activate nil bystander antibiotics in partnership with masked fluorophores for imaging purposes. It has previously been shown that antibiotics can be applied to a nitroreductase under control of a tissue-specific promoter in a transgenic model organism, enabling controlled ablation of that tissue at precise stages of development. However, direct imaging of the nitroreductase location and activity, by application of masked fluorophore probes prior to ablation, has not previously been explored.  During the course of this work, several promising combinations of nitroreductases that exhibit opposing specificities for certain combinations of masked fluorophores and nil-bystander antibiotics were identified through screening in bacterial systems. In general, these results were found to translate effectively into eukaryotic cell lines. Pairs of nitroreductases that have opposite specificities for two different antibiotic substrates offer potential for the multiplexed ablation of either (or both) of two different labelled tissues in the same transgenic model organism, according to the substrate(s) administered to that organism.  Throughout this screening process, a nitroaromatic substrate (niclosamide) was identified that is, uniquely, initially toxic to Escherichia coli but becomes non-toxic upon reduction of the nitro substituent. Using niclosamide, a novel strategy with potential for identification of new nitroreductases, as well as selection-based directed evolution to improve desired activities, was explored.</p>

scholarly journals The characterisation and application of bacterial nitroreductase enzymes

2021 ◽  
Author(s):  
◽  
Sarah Condon
Keyword(s):  
New Technologies ◽  
Active Form ◽  
Model Organism ◽  
Eukaryotic Cell ◽  
Screening Process ◽  
Enzyme Prodrug Therapy ◽  
Transgenic Model ◽  
Cell Ablation ◽  
Anti Cancer ◽  
Safety Features

<p>Cancer is an increasing global concern, with the number of people diagnosed growing rapidly each year. Gene directed enzyme prodrug therapy (GDEPT) is emerging as a front-runner of new technologies that seek to combat the growing number of cases. One developing approach to GDEPT involves the use of bacterial nitroreductase enzymes to reduce prodrug substrates, which, upon reduction to their active form, are toxic to cancer cells through DNA crosslinking.  Nitroreductases have the ability to activate a variety of nitro-quenched compounds, not only anti-cancer prodrugs, but also nil bystander antibiotics and masked fluorophores, through the reduction of strongly electron-withdrawing nitro substituents on aromatic rings. My research initially sought to exploit this capability by partnering nitroreductases with nil bystander antibiotics for targeted cell ablation, as a component of a larger gene directed enzyme prodrug therapy project. This has potential to provide important safety features for removal of viral and bacterial vectors following anti-cancer gene therapy.  From this, the main focus evolved into utilising nitroreductase enzymes for targeted cell ablation for applications in developmental and regenerative biology. This exploited the ability of nitroreductases to activate nil bystander antibiotics in partnership with masked fluorophores for imaging purposes. It has previously been shown that antibiotics can be applied to a nitroreductase under control of a tissue-specific promoter in a transgenic model organism, enabling controlled ablation of that tissue at precise stages of development. However, direct imaging of the nitroreductase location and activity, by application of masked fluorophore probes prior to ablation, has not previously been explored.  During the course of this work, several promising combinations of nitroreductases that exhibit opposing specificities for certain combinations of masked fluorophores and nil-bystander antibiotics were identified through screening in bacterial systems. In general, these results were found to translate effectively into eukaryotic cell lines. Pairs of nitroreductases that have opposite specificities for two different antibiotic substrates offer potential for the multiplexed ablation of either (or both) of two different labelled tissues in the same transgenic model organism, according to the substrate(s) administered to that organism.  Throughout this screening process, a nitroaromatic substrate (niclosamide) was identified that is, uniquely, initially toxic to Escherichia coli but becomes non-toxic upon reduction of the nitro substituent. Using niclosamide, a novel strategy with potential for identification of new nitroreductases, as well as selection-based directed evolution to improve desired activities, was explored.</p>

scholarly journals Delivery of systemic anti-cancer therapy during the COVID-19 pandemic

2021 ◽  
Author(s):  
Orla Fitzpatrick ◽  
Roisin Ní Dhonaill ◽  
Anna Linehan ◽  
Zac Coyne ◽  
Maeve Hennessy ◽  
...  
Keyword(s):  
Cancer Therapy ◽  
General Population ◽  
Cancer Care ◽  
Screening Process ◽  
Symptom Questionnaire ◽  
Treatment Review ◽  
Anti Cancer ◽  
System Changes ◽  
Hospital Visits ◽  
Telephone Screening

Abstract Background The first confirmed case of COVID-19 in Ireland was on February 29th 2020. From March until late April, the number of cases increased exponentially. The delivery of anti-cancer therapy during the COVID-19 pandemic was extremely challenging. In order to balance the benefits of continuing anti-cancer therapy with the associated increased hospital visits, combined with the risk of COVID-19 infection, we undertook a series of system changes in the delivery of cancer care. Methods Patients who attended our dayward over a 4-month period were included. Data were obtained from patient and chemotherapy prescribing records. Patients were screened for symptoms of COVID-19 at two separate timepoints: prior to their visit via telephone, and using a symptom questionnaire on arrival at the hospital. If patients displayed COVID-19 symptoms, they were isolated and a viral swab arranged. Results A total of 456 patients attended from January 1st to April 30th. The numbers of visits from January to April were 601, 586, 575, and 607, respectively. During this period, there were 2369 patient visits to the dayward and 1953 (82%) intravenous regimens administered. Of the 416 visits that did not lead to treatment, 114 (27%) were scheduled non-treatment review visits, 194 (47%) treatments were held due to disease-related illness, and 108 (26%) treatments were held due to treatment-related complications. Screening measurements were implemented on March 18th due to rising COVID-19 prevalence in the general population. Overall, 53 treatments were held due to the screening process: 19 patients (36%) elicited COVID-19 symptoms via telephone screening; 34 patients (64%) were symptomatic in our pre-assessment area and referred for swabs, of which 4 were positive. Those with a negative swab were rescheduled for chemotherapy the following week. Conclusions With careful systematic changes, safe and continued delivery of systemic anti-cancer therapy during the COVID-19 pandemic is possible.

scholarly journals Dynamic Membrane Localization of RNase Y in Bacillus subtilis

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Lina Hamouche ◽  
Cyrille Billaudeau ◽  
Anna Rocca ◽  
Arnaud Chastanet ◽  
Saravuth Ngo ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Active Form ◽  
Model Organism ◽  
Living Cells ◽  
Cell Periphery ◽  
Rnase E ◽  
Content Type ◽  
Endonucleolytic Cleavage ◽  
Rnase Y

ABSTRACT Metabolic turnover of mRNA is fundamental to the control of gene expression in all organisms, notably in fast-adapting prokaryotes. In many bacteria, RNase Y initiates global mRNA decay via an endonucleolytic cleavage, as shown in the Gram-positive model organism Bacillus subtilis. This enzyme is tethered to the inner cell membrane, a pseudocompartmentalization coherent with its task of initiating mRNA cleavage/maturation of mRNAs that are translated at the cell periphery. Here, we used total internal reflection fluorescence microscopy (TIRFm) and single-particle tracking (SPT) to visualize RNase Y and analyze its distribution and dynamics in living cells. We find that RNase Y diffuses rapidly at the membrane in the form of dynamic short-lived foci. Unlike RNase E, the major decay-initiating RNase in Escherichia coli, the formation of foci is not dependent on the presence of RNA substrates. On the contrary, RNase Y foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. The Y-complex of three proteins (YaaT, YlbF, and YmcA) has previously been shown to play an important role for RNase Y activity in vivo. We demonstrate that Y-complex mutations have an effect similar to but much stronger than that of depletion of RNA in increasing the number and size of RNase Y foci at the membrane. Our data suggest that the Y-complex shifts the assembly status of RNase Y toward fewer and smaller complexes, thereby increasing cleavage efficiency of complex substrates like polycistronic mRNAs. IMPORTANCE All living organisms must degrade mRNA to adapt gene expression to changing environments. In bacteria, initiation of mRNA decay generally occurs through an endonucleolytic cleavage. In the Gram-positive model organism Bacillus subtilis and probably many other bacteria, the key enzyme for this task is RNase Y, which is anchored at the inner cell membrane. While this pseudocompartmentalization appears coherent with translation occurring primarily at the cell periphery, our knowledge on the distribution and dynamics of RNase Y in living cells is very scarce. Here, we show that RNase Y moves rapidly along the membrane in the form of dynamic short-lived foci. These foci become more abundant and increase in size following transcription arrest, suggesting that they do not constitute the most active form of the nuclease. This contrasts with RNase E, the major decay-initiating RNase in E. coli, where it was shown that formation of foci is dependent on the presence of RNA substrates. We also show that a protein complex (Y-complex) known to influence the specificity of RNase Y activity in vivo is capable of shifting the assembly status of RNase Y toward fewer and smaller complexes. This highlights fundamental differences between RNase E- and RNase Y-based degradation machineries.

scholarly journals Evaluation of Cyclosaplin Efficacy Using a Silk Based 3D Tumor Model

Biomolecules ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 123 ◽  
Author(s):  
Abheepsa Mishra ◽  
Sourav K Mukhopadhyay ◽  
Satyahari Dey
Keyword(s):  
Breast Cancer ◽  
Cell Mass ◽  
3D Culture ◽  
Microscopic Analysis ◽  
Tumor Model ◽  
Rapid Screening ◽  
Screening Process ◽  
3D Microenvironment ◽  
Anti Cancer

Development of novel anti-cancer peptides requires a rapid screening process which can be accelerated by using appropriate in vitro tumor models. Breast carcinoma tissue is a three-dimensional (3D) microenvironment, which contains a hypoxic center surrounded by dense proliferative tissue. Biochemical clues provided by such a 3D cell mass cannot be recapitulated in conventional 2D culture systems. In this experiment, we evaluate the efficacy of the sandalwood peptide, cyclosaplin, on an established in vitro 3D silk breast cancer model using the invasive MDA-MB-231 cell line. The anti-proliferative effect of the peptide on the 3D silk tumor model is monitored by alamarBlue assay, with conventional 2D culture as control. The proliferation rate, glucose consumed, lactate dehydrogenase (LDH), and matrix metalloproteinase 9 (MMP-9) activity of human breast cancer cells are higher in 3D constructs compared to 2D. A higher concentration of drug is required to achieve 50% cell death in 3D culture than in 2D culture. The cyclosaplin treated MDA-MB-231 cells showed a significant decrease in MMP-9 activity in 3D constructs. Microscopic analysis revealed the formation of cell clusters evenly distributed in the scaffolds. The drug treated cells were less in number, smaller and showed unusual morphology. Overall, these findings indicate the role of cyclosaplin as a promising anti-cancer therapeutic.

scholarly journals Conditional Muller Cell Ablation Causes Independent Neuronal and Vascular Pathologies in a Novel Transgenic Model

2012 ◽  
Vol 32 (45) ◽  
pp. 15715-15727 ◽  
Author(s):  
W. Shen ◽  
M. Fruttiger ◽  
L. Zhu ◽  
S. H. Chung ◽  
N. L. Barnett ◽  
...  
Keyword(s):  
Müller Cell ◽  
Transgenic Model ◽  
Cell Ablation ◽  
Muller Cell

scholarly journals Vitamin D receptor as a target for breast cancer therapy

2017 ◽  
Vol 24 (4) ◽  
pp. 181-195 ◽  
Author(s):  
Alyson Murray ◽  
Stephen F Madden ◽  
Naoise C Synnott ◽  
Rut Klinger ◽  
Darran O'Connor ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Vitamin D ◽  
Cell Lines ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Active Form ◽  
Cancer Cell Line ◽  
Breast Cancer Cell Lines ◽  
Negative Cell ◽  
Anti Cancer

Considerable epidemiological evidence suggests that high levels of circulating vitamin D (VD) are associated with a decreased incidence and increased survival from cancer, i.e., VD may possess anti-cancer properties. The aim of this investigation was therefore to investigate the anti-cancer potential of a low calcaemic vitamin D analogue, i.e., inecalcitol and compare it with the active form of vitamin D, i.e., calcitriol, in a panel of breast cancer cell lines (n = 15). Using the MTT assay, IC50concentrations for response to calcitriol varied from 0.12 µM to >20 µM, whereas those for inecalcitol were significantly lower, ranging from 2.5 nM to 63 nM (P = 0.001). Sensitivity to calcitriol and inecalcitol was higher in VD receptor (VDR)-positive compared to VDR-negative cell lines (P = 0.0007 and 0.0080, respectively) and in ER-positive compared to ER-negative cell lines (P = 0.043 and 0.005, respectively). Using RNA-seq analysis, substantial but not complete overlap was found between genes differentially regulated by calcitriol and inecalcitol. In particular, significantly enriched gene ontology terms such as cell surface signalling and cell communication were found after treatment with inecalcitol but not with calcitriol. In contrast, ossification and bone morphogenesis were found significantly enriched after treatment with calcitriol but not with inecalcitol. Our preclinical results suggest that calcitriol and inecalcitol can inhibit breast cancer cell line growth, especially in cells expressing ER and VDR. As inecalcitol is significantly more potent than calcitriol and has low calcaemic potential, it should be further investigated for the treatment of breast cancer.

scholarly journals Resveratrol: A Pleiotropic Phytoconstituent

2020 ◽  
Vol 11 (SPL4) ◽  
pp. 1473-1479
Author(s):  
Prashant N. Amale ◽  
Manish P. Deshmukh ◽  
Ashish B. Budhrani ◽  
Shilpa A. Deshpande
Keyword(s):  
Nitric Oxide ◽  
Active Form ◽  
Global Market ◽  
Science Literature ◽  
Glutathione S Transferase ◽  
Reactive Protein ◽  
Anti Cancer ◽  
Web Science ◽  
Hepatoprotective Drug ◽  
Inducible Nitric Oxide

Resveratrol (RSV) is a plant polyphenol or phytolexin phytoconstituents obtained from the grapes, berries, peanut, and wine. RSV is obtained from natural source and regarded as safe, effective, and hepatoprotective drug with no other serious organ toxicities are reported yet. This property of RSV makes it advantageous over the allopathic medicine having symptomatic cure and plethora of adverse effects. It’s a cheap and widely available phytoconstituent approved in the global market in the active form as trans-resveratrol. It has multiple pharmacological actions including, analgesic, anti-inflammatory, anti-anxiety, anti-parkinsonian, anti-alzheimers, antioxidant, antidepressant, anti-cancer, anti-diabetic, anti-atherosclerotic effects. These effects are mediated via modulation of diverse underlying endogenous molecules like reactive oxygen species, nitric oxide, malonaldehyde, neutrophil, sirtulin, cyclo-oxygenase, inducible nitric oxide syntheses, superoxide dismutase, catalase, glutathione s-transferase, alpha-secretase, metalloproteinases, C-reactive protein, dopamine, nor-adrenaline, serotonin, cytokines (interleukins), nuclear factor kappa, signal transducer activator of transcription, brain derived neural nactor, neuropeptide, hypothalamo-pitutary axis, astroglia, mitochondrial dysfunction, glutamate, adrenergic, cholinergic, opioidergic, and purinergic receptors. Researchers are trying to explore its additional health benefits and preparing new analogues for better survival in the field. Present review will help to enlighten the multi-target pleiotropic pharmacological nature of a RSV in relation to the variety of the molecular targets modulation through extensive web science literature survey.

scholarly journals The role of vitamin D3 in signaling pathways – potential anticancer properties of calcitriol and its analogues

2019 ◽  
Vol 73 ◽  
pp. 920-936
Author(s):  
Olga Wiecheć
Keyword(s):  
Vitamin D ◽  
Cancer Cells ◽  
Vitamin D3 ◽  
Active Form ◽  
Signalling Pathways ◽  
Active Metabolites ◽  
Anticancer Properties ◽  
Anti Cancer ◽  
Worse Prognosis

Vitamin D, for many years after the discovery, primarily was associated with bone metabolic processes. Currently, many studies indicate its beneficial effect in the prevention and treatment of many diseases, including cancer. However, deficiency of vitamin D is associated with greater tendency to get sick and worse prognosis in treatment, especially cancer. Calcitriol, an active form of vitamin D (1.25(OH)2D3) and its analogues have a pleiotropic activity, including anti-cancer properties. Many studies indicate, that the active forms of vitamin D3 may show anti-proliferative effects in cancer cells by inhibiting the cell cycle, inducing differentiation or leading to apoptosis and enhancing autophagy. Also, extremely important are the possibilities of reducing the invasiveness of tumours through the influence on angiogenesis or adhesion and others. Especially, the anti-cancer role of vitamin D3 is suggested in the case of tumors whose cells express VDR receptors. Interestingly, many cancer cells not only express the VDR receptors, but also due to the expression of CYP27B1 and CYP24A hydroxylases, they can regulate metabolism of calcitriol. Many of the studies using vitamin D3 show that calcitriol and its analogues, due to the influence on cancer cells, can play promising roles in anticancer therapies. Consider the broad pleiotropism of the action of active metabolites of vitamin D3 and the development of research in this field, the current work presents the effect of active forms of vitamin D on some signalling pathways and the regulation of selected proteins in various cancers.

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