The recent discovery of a hydrolytic enzyme, IsPETase, that can deconstruct poly(ethylene) tereph... more The recent discovery of a hydrolytic enzyme, IsPETase, that can deconstruct poly(ethylene) terephthalate (PET), has sparked great interest in biocatalytic approaches to recycle plastics. Realisation of commercial utility will require the development of robust engineered enzymes that meet the demands of industrial processes. Although rationally engineered variants of PETases have been reported, enzymes that have been experimentally optimised through iterative rounds of directed evolution - the go-to method for engineering industrially useful biocatalysts – have not yet been described. Here, we report the development and implementation of an automated, high-throughput directed evolution platform for engineering polymer degrading enzymes. Evaluation of >13,000 IsPETase variants, applying catalytic activity at elevated temperatures as a primary selection pressure, afforded a HotPETase variant with 21 mutations that has a melting temperature of 82.5C and can therefore operate near or...
Alzheimer's disease (AD) is characterized by the presence of βamyloid plaques (Aβ) and neurofibri... more Alzheimer's disease (AD) is characterized by the presence of βamyloid plaques (Aβ) and neurofibrillary tangles (NFTs) in the brain. The prevalence of the disease is increasing and is expected to reach 141 million cases by 2050. Despite the risk factors associated with the disease, there is no known causative agent for AD. Clinical trials with many drugs have failed over the years, and no therapeutic has been approved for AD. There is increasing evidence that pathogens are found in the brains of AD patients and controls, such as human herpes simplex virus-1 (HSV-1). Given the lack of a human model, the route for pathogen entry into the brain remains open for scrutiny and may include entry via a disturbed blood−brain barrier or the olfactory nasal route. Many factors can contribute to the pathogenicity of HSV-1, such as the ability of HSV-1 to remain latent, tau protein phosphorylation, increased accumulation of Aβ in vivo and in vitro, and repeated cycle of reactivation if immunocompromised. Intriguingly, valacyclovir, a widely used drug for the treatment of HSV-1 and HSV-2 infection, has shown patient improvement in cognition compared to controls in AD clinical studies. We discuss the potential role of HSV-1 in AD pathogenesis and argue for further studies to investigate this relationship.
Directed evolution requires the creation of genetic diversity and subsequent screening or selecti... more Directed evolution requires the creation of genetic diversity and subsequent screening or selection for improved variants. For DNA mutagenesis, conventional sitedirected methods implicitly utilize the Boolean AND operator (creating all mutations simultaneously), producing a combinatorial explosion in the number of genetic variants as the number of mutations increases. We introduce GeneORator, a novel strategy for creating DNA libraries based on the Boolean logical OR operator. Here, a single library is divided into many subsets, each containing different combinations of the desired mutations. Consequently, the effect of adding more mutations on the number of genetic combinations is additive (Boolean OR logic) and not exponential (AND logic). We demonstrate this strategy with large-scale mutagenesis studies, using monoamine oxidase-N (Aspergillus niger) as the exemplar target. First, we mutated every residue in the secondary structurecontaining regions (276 out of a total 495 amino acids) to screen for improvements in k cat. Second, combinatorial OR-type libraries permitted screening of diverse mutation combinations in the enzyme active site to detect activity toward novel substrates. In both examples, OR-type libraries effectively reduced the number of variants searched up to 10 10-fold, dramatically reducing the screening effort required to discover variants with improved and/or novel activity. Importantly, this approach enables the screening of a greater diversity of mutation combinations, accessing a larger area of a protein's sequence space. ORtype libraries can be applied to any biological engineering objective requiring DNA mutagenesis, and the approach has wide ranging applications in, for example, enzyme engineering, antibody engineering, and synthetic biology.
Endoplasmic reticulum (ER) chaperone Prolyl 4-hydroxylase, beta polypeptide (P4HB) has previously... more Endoplasmic reticulum (ER) chaperone Prolyl 4-hydroxylase, beta polypeptide (P4HB) has previously been identified as a novel target for chemoresistance in glioblastoma multiforme (GBM). Yet its functional roles in glioma carcinogenesis remain elusive. In clinical analysis using human glioma specimens and Gene Expression Omnibus (GEO) profiles, we found that aberrant expression of P4HB was correlated with high-grade malignancy and an angiogenic phenotype in glioma. Furthermore, P4HB upregulation conferred malignant characteristics including proliferation, invasion, migration and angiogenesis in vitro, and increased tumor growth in vivo via the mitogen-activated protein kinase (MAPK) signaling pathway. Pathway analysis suggested genetic and pharmacologic inhibition of P4HB suppressed MAPK expression and its downstream targets were involved in angiogenesis and invasion. This is the first study that demonstrates the oncogenic roles of P4HB and its underlying mechanism in glioma. Since t...
Journal of the Royal Society, Interface, Mar 1, 2017
The theory of computer science is based around universal Turing machines (UTMs): abstract machine... more The theory of computer science is based around universal Turing machines (UTMs): abstract machines able to execute all possible algorithms. Modern digital computers are physical embodiments of classical UTMs. For the most important class of problem in computer science, non-deterministic polynomial complete problems, non-deterministic UTMs (NUTMs) are theoretically exponentially faster than both classical UTMs and quantum mechanical UTMs (QUTMs). However, no attempt has previously been made to build an NUTM, and their construction has been regarded as impossible. Here, we demonstrate the first physical design of an NUTM. This design is based on Thue string rewriting systems, and thereby avoids the limitations of most previous DNA computing schemes: all the computation is local (simple edits to strings) so there is no need for communication, and there is no need to order operations. The design exploits DNA's ability to replicate to execute an exponential number of computational pa...
Single cell transfection techniques are essential to understand the heterogeneity between cells. ... more Single cell transfection techniques are essential to understand the heterogeneity between cells. We have developed an integrated electrowetting nanoinjector (INENI) to transfect single cells. The high transfection efficiency, controlled dosage delivery and ease of INENI fabrication promote the widespread application of the INENI in cell transfection assays.
Integrative biology : quantitative biosciences from nano to macro, May 16, 2016
Patient-specific simulations, efficient parametric analyses, and the study of complex processes t... more Patient-specific simulations, efficient parametric analyses, and the study of complex processes that are otherwise experimentally intractable are facilitated through the use of Computational Fluid Dynamics (CFD) to study biological flows. This review discusses various CFD methodologies that have been applied across different biological scales, from cell to organ level. Through this discussion the lattice Boltzmann method (LBM) is highlighted as an emerging technique capable of efficiently simulating fluid problems across the midrange of scales; providing a practical analytical tool compared to methods more attuned to the extremities of scale. Furthermore, the merits of the LBM are highlighted through examples of previous applications and suggestions for future research are made. The review focusses on applications in the midrange bracket, such as cell-cell interactions, the microcirculation, and microfluidic devices; wherein the inherent mesoscale nature of the LBM renders it well s...
This review summarises the screening methods from hepatic ultrasonography to serological biomarke... more This review summarises the screening methods from hepatic ultrasonography to serological biomarkers for early detection of liver cancer and focuses on evaluation of biomarkers ability. The development of novel biomarkers according to the 5-phase program defined by the Early Detection Research Network (EDRN) is also outlined in this review.
The multifunctional protein-tissue inhibitor of metalloproteinases-1 (TIMP-1)-has been associated... more The multifunctional protein-tissue inhibitor of metalloproteinases-1 (TIMP-1)-has been associated with poor prognosis in several types of cancers including glioblastomas. Glioblastomas are the most common and malignant primary brain tumor known for being highly invasive and resistant to therapy. New treatment strategies are continuously being explored and currently vascular endothelial growth factor (VEGF) inhibitors administered in combination with Irinotecan is the most promising second line treatment. TIMP-1 has been associated with decreased response to chemotherapy in breast and colorectal cancer and especially the family of topoisomerase (TOP) inhibitors, such as Irinotecan, has been suggested to be affected by TIMP-1. In the present study, we investigated whether a high TIMP-1 expression in glioblastoma cells played a role in TOP inhibitor resistance. We established two TIMP-1 over-expressing cell lines and evaluated the sensitivity towards the TOP1 inhibitor SN-38 and the TOP2 inhibitor Epirubicin using a viability and a cytotoxicity assay. In addition, we investigated the invasive features of the cells in a brain slice culture model as well as in an orthotopic xenograft model. The results showed that TIMP-1 over-expressing U87MG cell line sub-clones were significantly more resistant than the controls when exposed to SN-38 and Epirubicin. The same tendency was seen for the TIMP-1 over-expressing A172 sub-clones. No significant differences in invasion patterns were observed for TIMP-1 over-expressing sub-clones when compared to controls. In conclusion, the present study suggests that TIMP-1 over-expression reduces the effect of TOP inhibitors in the glioblastoma cell line U87MG. There was no significant effect of TIMP-1 over-expression on tumor cell invasion. The association found between TIMP-1 cellular levels and the effect of TOP inhibitors needs to be validated in clinical patient material.
The need for transparency and good practices in the qPCR literature Two surveys of over 1,700 pub... more The need for transparency and good practices in the qPCR literature Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.
Background During the last decade, investigations have focused on revealing genes or proteins tha... more Background During the last decade, investigations have focused on revealing genes or proteins that are involved in HCC carcinogenesis using either genetic or proteomic techniques. However, these studies are overshadowed by a lack of good internal reference standards. The need to identify "housekeeping" markers, whose expression is stable in various experimental and clinical conditions, is therefore of the utmost clinical relevance in quantitative studies. This is the first study employed 2-DE analysis to screen for potential reference markers and aims to correlate the abundance of these proteins with their level of transcript expression. Methods A Chinese cohort of 224 liver tissues samples (105 cancerous, 103 non-tumourous cirrhotic, and 16 normal) was profiled using 2-DE analysis. Expression of the potential reference markers was confirmed by western blot, immunohistochemistry and real-time quantitative PCR. geNorm algorithm was employed for gene stability measure of the...
PCR retains a pivotal role in making accessible marker nucleic acid sequences for ready analysis ... more PCR retains a pivotal role in making accessible marker nucleic acid sequences for ready analysis in cancer diagnosis. For certain cancers such as acute lymphoblastic leukaemia, the application of quantitative procedures to assess and subsequently direct therapy has given rise to the slowly maturing field of MRD (minimal residual disease) management. Although excellent protocols exist for performing these analyses, akin to all PCR procedures the limit of detection can vary markedly between laboratories. The present paper is an overview that describes how the analytical field relating to miniaturization is likely to identify the missing link that integrates sample processing with downstream PCR, analysis and eventual therapy. Miniaturized devices are suited to the multi-parallelized handling of defined numbers of cells, and PCR-based microfluidic procedures have become reasonably established. The integration of sample processing and PCR in microfluidic devices is beginning to offer re...
Background: The pathogenesis of many diseases is correlated to irregularity in vascular endotheli... more Background: The pathogenesis of many diseases is correlated to irregularity in vascular endothelial growth factor (VEGF) expression. Results from several association studies show that variation in the level of VEGF expression is related to polymorphic sequences within the VEGF gene. Additionally, there are many studies showing that some gene polymorphisms significantly influence the pharmacokinetics of immunosuppressive drugs. Objective: The aim of this study was to determine the influence of immunosuppressive drugs on VEGF production in individuals with different VEGF genotypes. Methods: ARMS-PCR was used to genotype VEGF polymorphisms at positions-1154 and-2578 within the promoter of VEGF gene. A VEGF-specific ELISA was used to determine the influence of immunosuppressive drugs on VEGF production in PBMCs of individuals with different VEGF genotypes. Results: Suppressive effect of mycophenolic acid was observed just in individuals with GG-1154/CC-2578, GG-1154/CA-2578 and GA-1154/CC-2578 haplotypes. Additionally, VEGF was significantly suppressed in all individuals after treatment with rapamycin except those who had AA-1154/CA-2578 and AA-1154/AA-2578 VEGF genotype combinations. Conclusion: Results of a recent study revealed that MMF treatment might be effective in preventing chronic renal rejection only in recipients with IL-10 high producer genotype. Additionally result of another study showed that CYP3A5 genotype markedly influences the pharmacokinetics of rapamycin in kidney transplant recipients. Therefore with regard to our results, different suppressive effect of mycophenolic acid and rapamycin on VEGF production might also be dependent on VEGF genotype.
, as demonstrated by the stability of the 100-ns simulation at 300 K started from the native NMR ... more , as demonstrated by the stability of the 100-ns simulation at 300 K started from the native NMR structure. But accurate inclusion of long-range electrostatic effects [U. Essmann et al., J. Chem. Phys. 103, 8577 (1995)] does provide an additional challenge for achieving high parallelism in the MD code. Work is in progress to include long-range electrostatic effects while still achieving a level of parallelism and speed comparable to that presented here.
Up until recently, it was believed that pharmaceutical drugs and their metabolites enter into the... more Up until recently, it was believed that pharmaceutical drugs and their metabolites enter into the cell to gain access to their targets via simple diffusion across the hydrophobic lipid cellular membrane, at a rate which is based on their lipophilicity. An increasing amount of evidence indicates that the phospholipid bilayer-mediated drug diffusion is in fact negligible, and that drugs pass through cell membranes via proteinaceous membrane transporters or carriers which are normally used for the transportation of nutrients and intermediate metabolites. Drugs can be targeted to specific cells and tissues which express the relevant transporters, leading to the design of safe and efficacious treatments. Furthermore, transporter expression levels can be manipulated, systematically and in a high-throughput manner, allowing for considerable progress in determining which transporters are used by specific drugs. The ever-expanding field of miRNA therapeutics is not without its challenges, wi...
We recently introduced the Gini coefficient (GC) for assessing the expression variation of a part... more We recently introduced the Gini coefficient (GC) for assessing the expression variation of a particular gene in a dataset, as a means of selecting improved reference genes over the cohort (‘housekeeping genes’) typically used for normalisation in expression profiling studies. Those genes (transcripts) that we determined to be useable as reference genes differed greatly from previous suggestions based on hypothesis-driven approaches. A limitation of this initial study is that a single (albeit large) dataset was employed for both tissues and cell lines.We here extend this analysis to encompass seven other large datasets. Although their absolute values differ a little, the Gini values and median expression levels of the various genes are well correlated with each other between the various cell line datasets, implying that our original choice of the more ubiquitously expressed low-Gini-coefficient genes was indeed sound. In tissues, the Gini values and median expression levels of genes ...
We used high-throughput flow cytometry to assess the ability of individual gene knockout strains ... more We used high-throughput flow cytometry to assess the ability of individual gene knockout strains ofE colito take up two membrane-permeable, cationic fluorescent dyes, viz the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and β-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by say a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in both directions (increased or decreased); knockouts in known influx and efflux transporters behaved as expected, giving confidence in the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function (‘y-genes’). Similarly, several overexpression variants...
Improving enzymes by directed evolution requires the navigation of very large search spaces; we s... more Improving enzymes by directed evolution requires the navigation of very large search spaces; we survey how to do this intelligently.
The recent discovery of a hydrolytic enzyme, IsPETase, that can deconstruct poly(ethylene) tereph... more The recent discovery of a hydrolytic enzyme, IsPETase, that can deconstruct poly(ethylene) terephthalate (PET), has sparked great interest in biocatalytic approaches to recycle plastics. Realisation of commercial utility will require the development of robust engineered enzymes that meet the demands of industrial processes. Although rationally engineered variants of PETases have been reported, enzymes that have been experimentally optimised through iterative rounds of directed evolution - the go-to method for engineering industrially useful biocatalysts – have not yet been described. Here, we report the development and implementation of an automated, high-throughput directed evolution platform for engineering polymer degrading enzymes. Evaluation of >13,000 IsPETase variants, applying catalytic activity at elevated temperatures as a primary selection pressure, afforded a HotPETase variant with 21 mutations that has a melting temperature of 82.5C and can therefore operate near or...
Alzheimer's disease (AD) is characterized by the presence of βamyloid plaques (Aβ) and neurofibri... more Alzheimer's disease (AD) is characterized by the presence of βamyloid plaques (Aβ) and neurofibrillary tangles (NFTs) in the brain. The prevalence of the disease is increasing and is expected to reach 141 million cases by 2050. Despite the risk factors associated with the disease, there is no known causative agent for AD. Clinical trials with many drugs have failed over the years, and no therapeutic has been approved for AD. There is increasing evidence that pathogens are found in the brains of AD patients and controls, such as human herpes simplex virus-1 (HSV-1). Given the lack of a human model, the route for pathogen entry into the brain remains open for scrutiny and may include entry via a disturbed blood−brain barrier or the olfactory nasal route. Many factors can contribute to the pathogenicity of HSV-1, such as the ability of HSV-1 to remain latent, tau protein phosphorylation, increased accumulation of Aβ in vivo and in vitro, and repeated cycle of reactivation if immunocompromised. Intriguingly, valacyclovir, a widely used drug for the treatment of HSV-1 and HSV-2 infection, has shown patient improvement in cognition compared to controls in AD clinical studies. We discuss the potential role of HSV-1 in AD pathogenesis and argue for further studies to investigate this relationship.
Directed evolution requires the creation of genetic diversity and subsequent screening or selecti... more Directed evolution requires the creation of genetic diversity and subsequent screening or selection for improved variants. For DNA mutagenesis, conventional sitedirected methods implicitly utilize the Boolean AND operator (creating all mutations simultaneously), producing a combinatorial explosion in the number of genetic variants as the number of mutations increases. We introduce GeneORator, a novel strategy for creating DNA libraries based on the Boolean logical OR operator. Here, a single library is divided into many subsets, each containing different combinations of the desired mutations. Consequently, the effect of adding more mutations on the number of genetic combinations is additive (Boolean OR logic) and not exponential (AND logic). We demonstrate this strategy with large-scale mutagenesis studies, using monoamine oxidase-N (Aspergillus niger) as the exemplar target. First, we mutated every residue in the secondary structurecontaining regions (276 out of a total 495 amino acids) to screen for improvements in k cat. Second, combinatorial OR-type libraries permitted screening of diverse mutation combinations in the enzyme active site to detect activity toward novel substrates. In both examples, OR-type libraries effectively reduced the number of variants searched up to 10 10-fold, dramatically reducing the screening effort required to discover variants with improved and/or novel activity. Importantly, this approach enables the screening of a greater diversity of mutation combinations, accessing a larger area of a protein's sequence space. ORtype libraries can be applied to any biological engineering objective requiring DNA mutagenesis, and the approach has wide ranging applications in, for example, enzyme engineering, antibody engineering, and synthetic biology.
Endoplasmic reticulum (ER) chaperone Prolyl 4-hydroxylase, beta polypeptide (P4HB) has previously... more Endoplasmic reticulum (ER) chaperone Prolyl 4-hydroxylase, beta polypeptide (P4HB) has previously been identified as a novel target for chemoresistance in glioblastoma multiforme (GBM). Yet its functional roles in glioma carcinogenesis remain elusive. In clinical analysis using human glioma specimens and Gene Expression Omnibus (GEO) profiles, we found that aberrant expression of P4HB was correlated with high-grade malignancy and an angiogenic phenotype in glioma. Furthermore, P4HB upregulation conferred malignant characteristics including proliferation, invasion, migration and angiogenesis in vitro, and increased tumor growth in vivo via the mitogen-activated protein kinase (MAPK) signaling pathway. Pathway analysis suggested genetic and pharmacologic inhibition of P4HB suppressed MAPK expression and its downstream targets were involved in angiogenesis and invasion. This is the first study that demonstrates the oncogenic roles of P4HB and its underlying mechanism in glioma. Since t...
Journal of the Royal Society, Interface, Mar 1, 2017
The theory of computer science is based around universal Turing machines (UTMs): abstract machine... more The theory of computer science is based around universal Turing machines (UTMs): abstract machines able to execute all possible algorithms. Modern digital computers are physical embodiments of classical UTMs. For the most important class of problem in computer science, non-deterministic polynomial complete problems, non-deterministic UTMs (NUTMs) are theoretically exponentially faster than both classical UTMs and quantum mechanical UTMs (QUTMs). However, no attempt has previously been made to build an NUTM, and their construction has been regarded as impossible. Here, we demonstrate the first physical design of an NUTM. This design is based on Thue string rewriting systems, and thereby avoids the limitations of most previous DNA computing schemes: all the computation is local (simple edits to strings) so there is no need for communication, and there is no need to order operations. The design exploits DNA's ability to replicate to execute an exponential number of computational pa...
Single cell transfection techniques are essential to understand the heterogeneity between cells. ... more Single cell transfection techniques are essential to understand the heterogeneity between cells. We have developed an integrated electrowetting nanoinjector (INENI) to transfect single cells. The high transfection efficiency, controlled dosage delivery and ease of INENI fabrication promote the widespread application of the INENI in cell transfection assays.
Integrative biology : quantitative biosciences from nano to macro, May 16, 2016
Patient-specific simulations, efficient parametric analyses, and the study of complex processes t... more Patient-specific simulations, efficient parametric analyses, and the study of complex processes that are otherwise experimentally intractable are facilitated through the use of Computational Fluid Dynamics (CFD) to study biological flows. This review discusses various CFD methodologies that have been applied across different biological scales, from cell to organ level. Through this discussion the lattice Boltzmann method (LBM) is highlighted as an emerging technique capable of efficiently simulating fluid problems across the midrange of scales; providing a practical analytical tool compared to methods more attuned to the extremities of scale. Furthermore, the merits of the LBM are highlighted through examples of previous applications and suggestions for future research are made. The review focusses on applications in the midrange bracket, such as cell-cell interactions, the microcirculation, and microfluidic devices; wherein the inherent mesoscale nature of the LBM renders it well s...
This review summarises the screening methods from hepatic ultrasonography to serological biomarke... more This review summarises the screening methods from hepatic ultrasonography to serological biomarkers for early detection of liver cancer and focuses on evaluation of biomarkers ability. The development of novel biomarkers according to the 5-phase program defined by the Early Detection Research Network (EDRN) is also outlined in this review.
The multifunctional protein-tissue inhibitor of metalloproteinases-1 (TIMP-1)-has been associated... more The multifunctional protein-tissue inhibitor of metalloproteinases-1 (TIMP-1)-has been associated with poor prognosis in several types of cancers including glioblastomas. Glioblastomas are the most common and malignant primary brain tumor known for being highly invasive and resistant to therapy. New treatment strategies are continuously being explored and currently vascular endothelial growth factor (VEGF) inhibitors administered in combination with Irinotecan is the most promising second line treatment. TIMP-1 has been associated with decreased response to chemotherapy in breast and colorectal cancer and especially the family of topoisomerase (TOP) inhibitors, such as Irinotecan, has been suggested to be affected by TIMP-1. In the present study, we investigated whether a high TIMP-1 expression in glioblastoma cells played a role in TOP inhibitor resistance. We established two TIMP-1 over-expressing cell lines and evaluated the sensitivity towards the TOP1 inhibitor SN-38 and the TOP2 inhibitor Epirubicin using a viability and a cytotoxicity assay. In addition, we investigated the invasive features of the cells in a brain slice culture model as well as in an orthotopic xenograft model. The results showed that TIMP-1 over-expressing U87MG cell line sub-clones were significantly more resistant than the controls when exposed to SN-38 and Epirubicin. The same tendency was seen for the TIMP-1 over-expressing A172 sub-clones. No significant differences in invasion patterns were observed for TIMP-1 over-expressing sub-clones when compared to controls. In conclusion, the present study suggests that TIMP-1 over-expression reduces the effect of TOP inhibitors in the glioblastoma cell line U87MG. There was no significant effect of TIMP-1 over-expression on tumor cell invasion. The association found between TIMP-1 cellular levels and the effect of TOP inhibitors needs to be validated in clinical patient material.
The need for transparency and good practices in the qPCR literature Two surveys of over 1,700 pub... more The need for transparency and good practices in the qPCR literature Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.
Background During the last decade, investigations have focused on revealing genes or proteins tha... more Background During the last decade, investigations have focused on revealing genes or proteins that are involved in HCC carcinogenesis using either genetic or proteomic techniques. However, these studies are overshadowed by a lack of good internal reference standards. The need to identify "housekeeping" markers, whose expression is stable in various experimental and clinical conditions, is therefore of the utmost clinical relevance in quantitative studies. This is the first study employed 2-DE analysis to screen for potential reference markers and aims to correlate the abundance of these proteins with their level of transcript expression. Methods A Chinese cohort of 224 liver tissues samples (105 cancerous, 103 non-tumourous cirrhotic, and 16 normal) was profiled using 2-DE analysis. Expression of the potential reference markers was confirmed by western blot, immunohistochemistry and real-time quantitative PCR. geNorm algorithm was employed for gene stability measure of the...
PCR retains a pivotal role in making accessible marker nucleic acid sequences for ready analysis ... more PCR retains a pivotal role in making accessible marker nucleic acid sequences for ready analysis in cancer diagnosis. For certain cancers such as acute lymphoblastic leukaemia, the application of quantitative procedures to assess and subsequently direct therapy has given rise to the slowly maturing field of MRD (minimal residual disease) management. Although excellent protocols exist for performing these analyses, akin to all PCR procedures the limit of detection can vary markedly between laboratories. The present paper is an overview that describes how the analytical field relating to miniaturization is likely to identify the missing link that integrates sample processing with downstream PCR, analysis and eventual therapy. Miniaturized devices are suited to the multi-parallelized handling of defined numbers of cells, and PCR-based microfluidic procedures have become reasonably established. The integration of sample processing and PCR in microfluidic devices is beginning to offer re...
Background: The pathogenesis of many diseases is correlated to irregularity in vascular endotheli... more Background: The pathogenesis of many diseases is correlated to irregularity in vascular endothelial growth factor (VEGF) expression. Results from several association studies show that variation in the level of VEGF expression is related to polymorphic sequences within the VEGF gene. Additionally, there are many studies showing that some gene polymorphisms significantly influence the pharmacokinetics of immunosuppressive drugs. Objective: The aim of this study was to determine the influence of immunosuppressive drugs on VEGF production in individuals with different VEGF genotypes. Methods: ARMS-PCR was used to genotype VEGF polymorphisms at positions-1154 and-2578 within the promoter of VEGF gene. A VEGF-specific ELISA was used to determine the influence of immunosuppressive drugs on VEGF production in PBMCs of individuals with different VEGF genotypes. Results: Suppressive effect of mycophenolic acid was observed just in individuals with GG-1154/CC-2578, GG-1154/CA-2578 and GA-1154/CC-2578 haplotypes. Additionally, VEGF was significantly suppressed in all individuals after treatment with rapamycin except those who had AA-1154/CA-2578 and AA-1154/AA-2578 VEGF genotype combinations. Conclusion: Results of a recent study revealed that MMF treatment might be effective in preventing chronic renal rejection only in recipients with IL-10 high producer genotype. Additionally result of another study showed that CYP3A5 genotype markedly influences the pharmacokinetics of rapamycin in kidney transplant recipients. Therefore with regard to our results, different suppressive effect of mycophenolic acid and rapamycin on VEGF production might also be dependent on VEGF genotype.
, as demonstrated by the stability of the 100-ns simulation at 300 K started from the native NMR ... more , as demonstrated by the stability of the 100-ns simulation at 300 K started from the native NMR structure. But accurate inclusion of long-range electrostatic effects [U. Essmann et al., J. Chem. Phys. 103, 8577 (1995)] does provide an additional challenge for achieving high parallelism in the MD code. Work is in progress to include long-range electrostatic effects while still achieving a level of parallelism and speed comparable to that presented here.
Up until recently, it was believed that pharmaceutical drugs and their metabolites enter into the... more Up until recently, it was believed that pharmaceutical drugs and their metabolites enter into the cell to gain access to their targets via simple diffusion across the hydrophobic lipid cellular membrane, at a rate which is based on their lipophilicity. An increasing amount of evidence indicates that the phospholipid bilayer-mediated drug diffusion is in fact negligible, and that drugs pass through cell membranes via proteinaceous membrane transporters or carriers which are normally used for the transportation of nutrients and intermediate metabolites. Drugs can be targeted to specific cells and tissues which express the relevant transporters, leading to the design of safe and efficacious treatments. Furthermore, transporter expression levels can be manipulated, systematically and in a high-throughput manner, allowing for considerable progress in determining which transporters are used by specific drugs. The ever-expanding field of miRNA therapeutics is not without its challenges, wi...
We recently introduced the Gini coefficient (GC) for assessing the expression variation of a part... more We recently introduced the Gini coefficient (GC) for assessing the expression variation of a particular gene in a dataset, as a means of selecting improved reference genes over the cohort (‘housekeeping genes’) typically used for normalisation in expression profiling studies. Those genes (transcripts) that we determined to be useable as reference genes differed greatly from previous suggestions based on hypothesis-driven approaches. A limitation of this initial study is that a single (albeit large) dataset was employed for both tissues and cell lines.We here extend this analysis to encompass seven other large datasets. Although their absolute values differ a little, the Gini values and median expression levels of the various genes are well correlated with each other between the various cell line datasets, implying that our original choice of the more ubiquitously expressed low-Gini-coefficient genes was indeed sound. In tissues, the Gini values and median expression levels of genes ...
We used high-throughput flow cytometry to assess the ability of individual gene knockout strains ... more We used high-throughput flow cytometry to assess the ability of individual gene knockout strains ofE colito take up two membrane-permeable, cationic fluorescent dyes, viz the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and β-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by say a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in both directions (increased or decreased); knockouts in known influx and efflux transporters behaved as expected, giving confidence in the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function (‘y-genes’). Similarly, several overexpression variants...
Improving enzymes by directed evolution requires the navigation of very large search spaces; we s... more Improving enzymes by directed evolution requires the navigation of very large search spaces; we survey how to do this intelligently.
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Papers by Philip Day