Tunable Release of Ions from Graphene Oxide Laminates for Sustained Antibacterial Activity in a Biomimetic Environment.

Silver has long been recognized for its potent antimicrobial properties, but achieving a slow and longer-term delivery of silver ions presents significant challenges. Previous efforts to control silver ion dosages have struggled to sustain release for extended periods in biomimetic environments, especially in the presence of complex proteins. This challenge is underscored by the absence of technology for sustaining antimicrobial activity, especially in the context of orthopedic implants where long-term efficacy, extending beyond 7 days, is essential. In this study, the tunable, slow, and longer-term release of silver ions from the two-dimensional (2D) nanocapillaries of graphene oxide (GO) laminates incorporated with silver ions (Ag-GO) for antimicrobial applications are successfully demonstrated. To closely mimic a physiologically relevant serum-based environment, a novel in vitro study model using 100% fetal bovine serum (FBS) is introduced as the test medium for microbiology, biocompatibility, and bioactivity studies. To emulate fluid circulation in a physiological environment, the in vitro studies are challenged with serum exchange protocols on different days. The findings show that the Ag-GO coating can sustainably release silver ions at a minimum dosage of 10 µg cm-2 day-1, providing an effective and sustained antimicrobial barrier for over ten days.

An untargeted metabolomics strategy to measure differences in metabolite uptake and excretion by mammalian cell lines.

INTRODUCTION: It is widely but erroneously believed that drugs get into cells by passing through the phospholipid bilayer portion of the plasma and other membranes. Much evidence shows, however, that this is not the case, and that drugs cross biomembranes by hitchhiking on transporters for other natural molecules to which these drugs are structurally similar. Untargeted metabolomics can provide a method for determining the differential uptake of such metabolites. OBJECTIVES: Blood serum contains many thousands of molecules and provides a convenient source of biologically relevant metabolites. Our objective was to detect and identify metabolites present in serum, but to also establish a method capable of measure their uptake and secretion by different cell lines. METHODS: We develop an untargeted LC-MS/MS method to detect a broad range of compounds present in human serum. We apply this to the analysis of the time course of the uptake and secretion of metabolites in serum by several human cell lines, by analysing changes in the serum that represents the extracellular phase (the 'exometabolome' or metabolic footprint). RESULTS: Our method measures some 4000-5000 metabolic features in both positive and negative electrospray ionisation modes. We show that the metabolic footprints of different cell lines differ greatly from each other. CONCLUSION: Our new, 15-min untargeted metabolome method allows for the robust and convenient measurement of differences in the uptake of serum compounds by cell lines following incubation in serum. This will enable future research to study these differences in multiple cell lines that will relate this to transporter expression, thereby advancing our knowledge of transporter substrates, both natural and xenobiotic compounds.

Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of microbial (re)growth.

Rapid changes in the number and flow cytometric behaviour of cells of E. coli taken from a stationary phase and inoculated into rich medium.Cells of E. coli were grown in LB medium, taken from a stationary phase of 2-4 h, and re-inoculated into fresh media at a concentration (105 ml-1 or lower) characteristic of bacteriuria. Flow cytometry was used to assess how quickly we could detect changes in cell size, number, membrane energization (using a carbocyanine dye) and DNA distribution. It transpired that while the lag phase observable macroscopically via bulk OD measurements could be as long as 4 h, the true lag phase could be less than 15-20 min, and was accompanied by many observable biochemical changes. Antibiotics to which the cells were sensitive affected these changes within 20 min of re-inoculation, providing the possibility of a very rapid antibiotic susceptibility test on a timescale compatible with a visit to a GP clinic. The strategy was applied successfully to genuine potential urinary tract infection (UTI) samples taken from a doctor's surgery. The methods developed could prove of considerable value in ensuring the correct prescription and thereby lowering the spread of antimicrobial resistance.

Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli.

BACKGROUND: It is widely believed that most xenobiotics cross biomembranes by diffusing through the phospholipid bilayer, and that the use of protein transporters is an occasional adjunct. According to an alternative view, phospholipid bilayer transport is negligible, and several different transporters may be involved in the uptake of an individual molecular type. We recognise here that the availability of gene knockout collections allows one to assess the contributions of all potential transporters, and flow cytometry based on fluorescence provides a convenient high-throughput assay for xenobiotic uptake in individual cells. RESULTS: We used high-throughput flow cytometry to assess the ability of individual gene knockout strains of E coli to take up two membrane-permeable, cationic fluorescent dyes, namely 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 ß-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by 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 either direction (increased or decreased); knockouts of known influx and efflux transporters behaved as expected, giving credence to the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function ('y-genes'). Similarly, several overexpression variants in the 'ASKA' collection had the anticipated, opposite effects. Similar results were obtained with SYBR Green (the range being approximately 69-fold). Although it too contains a benzothiazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains (although the membrane potential is presumably the same in each case). CONCLUSIONS: Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters of putatively broad and presently unknown specificity, and that the very large range between the 'lowest' and the 'highest' levels of uptake, even in knockouts of just single genes, implies strongly that phospholipid bilayer transport is indeed negligible. This work also casts serious doubt upon the use of such dyes as quantitative stains for representing either bioenergetic parameters or the amount of cellular DNA in unfixed cells (in vivo). By contrast, it opens up their potential use as transporter assay substrates in high-throughput screening.

Integrative analysis of rewired central metabolism in temozolomide resistant cells.

An authenticated U87MG clonal glioblastoma cell line was investigated to identify a sub-population of neurospheroidal (NSP) cells within the main epithelial population (U87MG). The NSP cells sorted using Fluorescence Assisted Cell Sorting (FACS) showed varied morphology, 30% lower growth rates, 40% higher IC50 values for temozolomide drug and could differentiate into the glial cell type (NDx). Metabolite profiling using HR-LCMS identified glucose, glutamine and serine in both populations and tryptophan only in U87MG as growth limiting substrates. Glycine, alanine, glutamate and proline were secreted by U87MG, however proline and glycine were re-utilized in NSP. Exo-metabolite profiling and phenotypic microarrays identified differential metabolism of primary carbon sources glucose and derived pyruvate for U87MG; glutamine and derived glutamate metabolism in NSP. Differential mRNA abundance of AKT1, PTEN, PIK3CA controlling metabolism, drug efflux, nutrient transport and epigenetic control MDM2 are potentially critical in shaping DNA methylation effects of temozolomide. Our study provides a new insight into the combined effect of these factors leading to temozolomide resistance in NSP.

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.

GeneGenie: optimized oligomer design for directed evolution.

GeneGenie, a new online tool available at http://www.gene-genie.org, is introduced to support the design and self-assembly of synthetic genes and constructs. GeneGenie allows for the design of oligonucleotide cohorts encoding the gene sequence optimized for expression in any suitable host through an intuitive, easy-to-use web interface. The tool ensures consistent oligomer overlapping melting temperatures, minimizes the likelihood of misannealing, optimizes codon usage for expression in a selected host, allows for specification of forward and reverse cloning sequences (for downstream ligation) and also provides support for mutagenesis or directed evolution studies. Directed evolution studies are enabled through the construction of variant libraries via the optional specification of 'variant codons', containing mixtures of bases, at any position. For example, specifying the variant codon TNT (where N is any nucleotide) will generate an equimolar mixture of the codons TAT, TCT, TGT and TTT at that position, encoding a mixture of the amino acids Tyr, Ser, Cys and Phe. This facility is demonstrated through the use of GeneGenie to develop and synthesize a library of enhanced green fluorescent protein variants.

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently.

The amino acid sequence of a protein affects both its structure and its function. Thus, the ability to modify the sequence, and hence the structure and activity, of individual proteins in a systematic way, opens up many opportunities, both scientifically and (as we focus on here) for exploitation in biocatalysis. Modern methods of synthetic biology, whereby increasingly large sequences of DNA can be synthesised de novo, allow an unprecedented ability to engineer proteins with novel functions. However, the number of possible proteins is far too large to test individually, so we need means for navigating the 'search space' of possible protein sequences efficiently and reliably in order to find desirable activities and other properties. Enzymologists distinguish binding (Kd) and catalytic (kcat) steps. In a similar way, judicious strategies have blended design (for binding, specificity and active site modelling) with the more empirical methods of classical directed evolution (DE) for improving kcat (where natural evolution rarely seeks the highest values), especially with regard to residues distant from the active site and where the functional linkages underpinning enzyme dynamics are both unknown and hard to predict. Epistasis (where the 'best' amino acid at one site depends on that or those at others) is a notable feature of directed evolution. The aim of this review is to highlight some of the approaches that are being developed to allow us to use directed evolution to improve enzyme properties, often dramatically. We note that directed evolution differs in a number of ways from natural evolution, including in particular the available mechanisms and the likely selection pressures. Thus, we stress the opportunities afforded by techniques that enable one to map sequence to (structure and) activity in silico, as an effective means of modelling and exploring protein landscapes. Because known landscapes may be assessed and reasoned about as a whole, simultaneously, this offers opportunities for protein improvement not readily available to natural evolution on rapid timescales. Intelligent landscape navigation, informed by sequence-activity relationships and coupled to the emerging methods of synthetic biology, offers scope for the development of novel biocatalysts that are both highly active and robust.

The role of microRNAs in the idiopathic inflammatory myopathies.

PURPOSE OF REVIEW: The idiopathic inflammatory myopathies (IIMs) are a group of rare autoimmune disorders characterized by skeletal muscle weakness and inflammation. MicroRNAs (miRNAs) regulate a wide range of developmental and physiological cellular processes. New approaches to investigating the mechanisms involved in IIM, such as investigating the role of miRNAs, are vital for the development of novel therapeutics and/or better diagnostic tools. RECENT FINDINGS: Identification of dysregulated miRNAs has led to a greater understanding of inflammation, muscle weakness/wasting and extra-muscular organ involvement in IIM. Up-regulation of immune-related miRNAs in muscle, for example, miR-155 and miR-146, is associated with autoimmunity, whereas down-regulation of myogenic miRNAs, including miR-1 and miR-206, is associated with inhibition of muscle regeneration. Disease mechanisms have been explored by altering in-vitro conditions and monitoring miRNA levels of interest, or, alternatively, changing miRNA levels and monitoring possible targets. For example, higher levels of cytokines appear to inhibit myogenic miRNAs in muscle and artificially reducing levels of miR-223 increases protein kinase C-epsilon (PKCε) levels in keratinocytes. SUMMARY: The exciting expansion of the miRNA field adds to our understanding of IIM pathogenesis and may provide future clinical potential either as diagnostic tools or as therapeutics via use of anti-miRNAs or synthetic miRNAs.

Microfluidic channel-assisted screening of hematopoietic malignancies.

A simple microfluidic fluorescence in situ hybridization (FISH) device allowing accurate analysis of interphase nuclei in 1 hr in narrow channels is presented. Photolithography and fluorosilicic acid etching were used to fabricate microfluidic channels (referred to as FISHing lines) that allowed analysis of 10 samples on a glass microscope slide 0.2 µl of sample volume was used to fill a micro-channel, which resembled a 250-fold reduction compared to conventional FISH. FISH signals were comparable to conventional FISH, with 50-fold less probe consumption and 10-fold less time. Cells were immobilized in single file in channels just exceeding the diameter of the cells, and were used for minimal residual disease (MRD) analysis. To test the micro-channels for application in FISH, MRD was simulated by mixing K562 cells (an established chronic myeloid leukemia cell line) carrying the BCR/ABL fusion gene across 1:1 to 1:1,000 Jurkat cells (an established acute lymphoblastic leukemia cell line). The limit of detection was seen to be 1:100 cells and 1:1,000 cells for FISHing lines and conventional FISH, respectively; however, the conventional method seemed to over-score the presence of K562 cells. This may in part be attributed to FISHing lines practically eliminating the chance of duplicate screening of cells and hastened the time of screening, enhancing scoring of all cells within the channels. This was compared to 1 in 500 cells on the slide being analyzed with the conventional FISH.

Shortened telomere length in patients with systemic lupus erythematosus.

OBJECTIVE: Patients with systemic lupus erythematosus (SLE) have a higher rate of premature death compared to the general population, suggesting a phenotype of premature senescence in SLE. Telomere length can be used to assess overall biologic aging. This study was undertaken to address the hypothesis that patients with SLE have reduced telomere length. METHODS: Telomere length was measured cross-sectionally in whole blood from SLE patients and age-matched healthy female controls, using real-time quantitative polymerase chain reaction. SLE-related and cardiovascular risk factors were assessed. RESULTS: We compared telomere length in 63 SLE patients and 63 matched controls with a median age of 50.8 years (interquartile range [IQR] 37-59 years) and 49.9 years (IQR 32-60 years), respectively. The median relative telomere length in SLE patients was 0.97 (IQR 0.47-1.57), compared to 1.53 (IQR 0.82-2.29) in controls (P = 0.0008). We then extended our cohort to measure telomere length in 164 SLE patients. Shorter telomere length was associated with Ro antibodies (ß ± SE -0.36 ± 0.16; P = 0.023), and longer telomere length was associated with steroid therapy (0.29 ± 0.14; P = 0.046). We also noted an association of longer telomere length with increasing body mass index (ß ± SE 0.07 ± 0.01; P < 0.0001) and tobacco smoking (0.64 ± 0.26; P = 0.016), as well as with the presence of carotid plaque (0.203 ± 0.177; P = 0.032). CONCLUSION: Telomere length is shortened in SLE patients compared to controls and does not appear to be a reflection of disease activity or immune cell turnover. Subsets of patients such as those positive for Ro antibodies may be particularly susceptible to premature biologic aging. The predictive value of telomere length as a biomarker of future risk of damage/mortality in SLE requires longitudinal evaluation.

Protein engineering enables a soakable crystal form of human CDK7 primed for high-throughput crystallography and structure-based drug design.

Cyclin dependent kinase 7 (CDK7) is an important therapeutic kinase best known for its dual role in cell cycle regulation and gene transcription. Here, we describe the application of protein engineering to generate constructs leading to high resolution crystal structures of human CDK7 in both active and inactive conformations. The active state of the kinase was crystallized by incorporation of an additional surface residue mutation (W132R) onto the double phosphomimetic mutant background (S164D and T170E) that yielded the inactive kinase structure. A novel back-soaking approach was developed to determine crystal structures of several clinical and pre-clinical inhibitors of this kinase, demonstrating the potential utility of the crystal system for structure-based drug design (SBDD). The crystal structures help to rationalize the mode of inhibition and the ligand selectivity profiles versus key anti-targets. The protein engineering approach described here illustrates a generally applicable strategy for structural enablement of challenging molecular targets.

Fragment-Based Discovery of Allosteric Inhibitors of SH2 Domain-Containing Protein Tyrosine Phosphatase-2 (SHP2).

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2. Structure-guided optimization, including several computational methods, led to the discovery of two structurally distinct series of SHP2 inhibitors binding to the previously reported allosteric tunnel binding site (Tunnel Site). One of these series was advanced to a low-nanomolar lead that inhibited tumor growth when dosed orally to mice bearing HCC827 xenografts. Furthermore, a third series of SHP2 inhibitors was discovered binding to a previously unreported site, lying at the interface of the C-terminal SH2 and catalytic domains.

Protein alterations associated with temozolomide resistance in subclones of human glioblastoma cell lines.

Temozolomide (TMZ) is the standard chemotherapeutic agent for human malignant glioma, but intrinsic or acquired chemoresistance represents a major obstacle to successful treatment of this highly lethal group of tumours. Obtaining better understanding of the molecular mechanisms underlying TMZ resistance in malignant glioma is important for the development of better treatment strategies. We have successfully established a passage control line (D54-C10) and resistant variants (D54-P5 and D54-P10) from the parental TMZ-sensitive malignant glioma cell line D54-C0. The resistant sub-cell lines showed alterations in cell morphology, enhanced cell adhesion, increased migration capacities, and cell cycle arrests. Proteomic analysis identified a set of proteins that showed gradual changes in expression according to their 50% inhibitory concentration (IC(50)). Successful validation was provided by transcript profiling in another malignant glioma cell line U87-MG and its resistant counterparts. Moreover, three of the identified proteins (vimentin, cathepsin D and prolyl 4-hydroxylase, beta polypeptide) were confirmed to be upregulated in high-grade glioma. Our data suggest that acquired TMZ resistance in human malignant glioma is associated with promotion of malignant phenotypes, and our reported molecular candidates may serve not only as markers of chemoresistance but also as potential therapeutic targets in the treatment of TMZ-resistant human malignant glioma, providing a platform for future investigations.

Crystal structure of human CDK4 in complex with a D-type cyclin.

The cyclin D1-cyclin-dependent kinase 4 (CDK4) complex is a key regulator of the transition through the G(1) phase of the cell cycle. Among the cyclin/CDKs, CDK4 and cyclin D1 are the most frequently activated by somatic genetic alterations in multiple tumor types. Thus, aberrant regulation of the CDK4/cyclin D1 pathway plays an essential role in oncogenesis; hence, CDK4 is a genetically validated therapeutic target. Although X-ray crystallographic structures have been determined for various CDK/cyclin complexes, CDK4/cyclin D1 has remained highly refractory to structure determination. Here, we report the crystal structure of CDK4 in complex with cyclin D1 at a resolution of 2.3 A. Although CDK4 is bound to cyclin D1 and has a phosphorylated T-loop, CDK4 is in an inactive conformation and the conformation of the heterodimer diverges from the previously known CDK/cyclin binary complexes, which suggests a unique mechanism for the process of CDK4 regulation and activation.

Perturbation of cell cycle expression in keloid fibroblast.

The pathogenesis of keloid formation is poorly understood. The fibroblasts in keloid patients continue to multiply even after initial wound repair and are characterized by a persistent dermal fibroproliferative reaction and excessive extracellular matrix production. Most studies concentrate on the type of collagen produced within keloids and the cytokines that dominate the disease. There have been considerably fewer studies in the expression of messenger RNA level in key cell cycle genes of the keloid fibroblast. The aim of this study was to measure the messenger RNA expression of the key regulators of cell cycle, cell cycle cyclins, and cyclin-dependent kinases, and their inhibitors.

Cell Surface Transporters and Novel Drug Developments.

Despite the numerous scientific and technological advances made within the last decade the attrition rates for new drug discovery remain as high as 95% for anticancer drugs. Recent drug development has been in part guided by Lipinski's Rule of 5 (Ro5) even though many approved drugs do not comply to these rules. With Covid-19 vaccine development strategy dramatically accelerating drug development perhaps it is timely to question the generic drug development process itself to find a more efficient, cost effective, and successful approach. It is widely believed that drugs permeate cells via two methods: phospholipid bilayer diffusion and carrier mediated transporters. However, emerging evidence suggests that carrier mediated transport may be the primary mechanism of drug uptake and not diffusion as long believed. Computational biology increasingly assists drug design to achieve desirable absorption, distribution, metabolism, elimination and toxicity (ADMET) properties. Perfecting drug entry into target cells as a prerequisite to intracellular drug action is a logical and compelling route and is expected to reduce drug attrition rates, particularly gaining favour amongst chronic lifelong therapeutics. Novel drug development is rapidly expanding from the utilisation of beyond the rule of five (bRo5) to pulsatile drug delivery systems and fragment based drug design. Utilising transporters as drug targets and advocating bRo5 molecules may be the solution to increasing drug specificity, reducing dosage and toxicity and thus revolutionising drug development. This review explores the development of cell surface transporter exploitation in drug development and the relationship with improved therapeutic index.

High-throughput droplet PCR.

The polymerase chain reaction has facilitated the ready analysis of nucleic acids. A next challenge requires the development of means to unravel the complexity of heterogeneous tissues. This has presented the task of producing massively parallelized quantitative nucleic acid data from the cellular constituents of tissues. The production of aqueous droplets in a two phase flow is shown to be readily and routinely facilitated by miniaturized fluidic devices. Droplets serve as ideal means to package a future generation of PCR, offering an enhanced handling potential by virtue of reactant containment, to concurrently eliminate both contamination and sample loss. This containment also enables the measurement of nucleic acids from populations of cells, or molecules by means of high throughput, single cell analysis. Details are provided for the production of a prototype micro-fluidic device which shows the production and stable flow of droplets which we suggest will be suitable for droplet-based continuous flow micro-fluidic PCR. Suggestions are also made as to the optimal fabrication techniques and the importance of device calibration.

Array-based evolution of DNA aptamers allows modelling of an explicit sequence-fitness landscape.

Mapping the landscape of possible macromolecular polymer sequences to their fitness in performing biological functions is a challenge across the biosciences. A paradigm is the case of aptamers, nucleic acids that can be selected to bind particular target molecules. We have characterized the sequence-fitness landscape for aptamers binding allophycocyanin (APC) protein via a novel Closed Loop Aptameric Directed Evolution (CLADE) approach. In contrast to the conventional SELEX methodology, selection and mutation of aptamer sequences was carried out in silico, with explicit fitness assays for 44,131 aptamers of known sequence using DNA microarrays in vitro. We capture the landscape using a predictive machine learning model linking sequence features and function and validate this model using 5500 entirely separate test sequences, which give a very high observed versus predicted correlation of 0.87. This approach reveals a complex sequence-fitness mapping, and hypotheses for the physical basis of aptameric binding; it also enables rapid design of novel aptamers with desired binding properties. We demonstrate an extension to the approach by incorporating prior knowledge into CLADE, resulting in some of the tightest binding sequences.

An Analysis of Mechanisms for Cellular Uptake of miRNAs to Enhance Drug Delivery and Efficacy in Cancer Chemoresistance.

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, with the most notable one being the safe and effective delivery of the miRNA mimic/antagonist safely to the target cell cytoplasm for attaining the desired clinical outcome, particularly in miRNA-based cancer therapeutics, due to the poor efficiency of neo-vascular systems revolting around the tumour site, brought about by tumour-induced angiogenesis. This acquisition of resistance to several types of anticancer drugs can be as a result of an upregulation of efflux transporters expression, which eject drugs from cells, hence lowering drug efficacy, resulting in multidrug resistance. In this article, the latest available data on human microRNAs has been reviewed, together with the most recently described mechanisms for miRNA uptake in cells, for future therapeutic enhancements against cancer chemoresistance.