Development of a Pain Management Protocol for a Paediatric Ward in the Gambia, West Africa

Despite recent advances in our understanding of paediatric pain and its management, pain continues to be undertreated globally, particularly in children and in low income countries. This article describes the development of a paediatric analgesia and sedation protocol, tailored to the specific setting of the Medical Research Council (MRC) paediatric ward in the Gambia, West Africa. An iterative process was used throughout development, with inputs from the medical literature, local providers, and pain experts, incorporated to ensure a safe, effective, and locally appropriate protocol. We demonstrate that evidence-based published guidelines, can and should be adapted to allow for optimal pain management given the resources and capabilities of specific health care settings. It is hoped that the process and protocol described here, will not only help to improve care on the MRC ward, but serve as an example to others working toward improving pain management in similar health care settings

A Three-Dimensional Model of Bacterial Biofilms and Its Use in Antimicrobial Susceptibility Testing

(1) Background: The discrepant antimicrobial susceptibility between planktonic and biofilm bacterial modes poses a problem for clinical microbiology laboratories and necessitates a relevant 3D experimental model allowing bacteria to grow in biofilm mode, in vitro, for use in anti-biofilm susceptibility testing. (2) Methods: This work develops a 3D biofilm model consisting of alginate beads containing S. aureus biofilm and encased within two thick layers of alginate matrix. The constructed model was placed on a thin Boyden chamber insert suspended on a 24-well culture plate containing the culture medium. The antibacterial activity of bacitracin and chlorhexidine digluconate (CD), either combined or separately, against 2D S. aureus culture was compared to that in the 3D biofilm model. Quantitative analysis and imaging analysis were performed by assessing the bacterial load within the matrix as well as measuring the optical density of the culture medium nourishing the matrix. (3) Results: The 3D biofilm model represented the typical complex characteristics of biofilm with greater insusceptibility to the tested antimicrobials than the 2D culture. Only bacitracin and CD in combination at 100× the concentration found to be successful against 2D culture were able to completely eliminate the 3D biofilm matrix. (4) Conclusions: The 3D biofilm model, designed to be more clinically relevant, exhibits higher antimicrobial insusceptibility than the 2D culture, demonstrating that the model might be useful for testing and discovering new antimicrobial therapies. The data also support the view that combination therapy might be the optimal approach to combat biofilm infections

Transduction‐Specific ATLAS Reveals a Cohort of Highly Active L 1 Retrotransposons in Human Populations

L ong IN terspersed E lement‐1 ( LINE ‐1 or L 1) retrotransposons are the only autonomously active transposable elements in the human genome. The average human genome contains ∼80–100 active L1s, but only a subset of these L1s are highly active or ‘hot’. Human L1s are closely related in sequence, making it difficult to decipher progenitor/offspring relationships using traditional phylogenetic methods. However, L1 m RNA s can sometimes bypass their own polyadenylation signal and instead utilize fortuitous polyadenylation signals in 3′ flanking genomic DNA . Retrotransposition of the resultant m RNA s then results in lineage specific sequence “tags” (i.e., 3′ transductions) that mark the descendants of active L1 progenitors. Here, we developed a method (Transduction‐Specific Amplification Typing of L1 Active Subfamilies or TS ‐ ATLAS ) that exploits L1 3′ transductions to identify active L1 lineages in a genome‐wide context. TS ‐ ATLAS enabled the characterization of a putative active progenitor of one L1 lineage that includes the disease causing L1 insertion L1 RP , and the identification of new retrotransposition events within two other “hot” L1 lineages. Intriguingly, the analysis of the newly discovered transduction lineage members suggests that L1 polyadenylation, even within a lineage, is highly stochastic. Thus, TS ‐ ATLAS provides a new tool to explore the dynamics of L1 lineage evolution and retrotransposon biology. Long INterspersed Element‐1 (L1) retrotransposons are the only independently mobile elements in the human genome. We developed Transduction‐Specific Amplification Typing of L1 Active Subfamilies (TS‐ATLAS), which utilizes L1‐transduced genomic sequences, to identify a subset of highly active L1s genome‐wide. TS‐ATLAS enabled the characterization of the putative progenitor of an active disease‐causing L1 lineage, and identified new retrotransposition events within two other “hot” L1 lineages.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98809/1/humu22327.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/98809/2/humu22327-sup-0001-si.pd

LINE-1 Retrotransposition Activity in Human Genomes

SummaryHighly active (i.e., “hot”) long interspersed element-1 (LINE-1 or L1) sequences comprise the bulk of retrotransposition activity in the human genome; however, the abundance of hot L1s in the human population remains largely unexplored. Here, we used a fosmid-based, paired-end DNA sequencing strategy to identify 68 full-length L1s that are differentially present among individuals but are absent from the human genome reference sequence. The majority of these L1s were highly active in a cultured cell retrotransposition assay. Genotyping 26 elements revealed that two L1s are only found in Africa and that two more are absent from the H952 subset of the Human Genome Diversity Panel. Therefore, these results suggest that hot L1s are more abundant in the human population than previously appreciated, and that ongoing L1 retrotransposition continues to be a major source of interindividual genetic variation

The influence of nanotexturing of poly(lactic-co-glycolic acid) films upon human ovarian cancer cell attachment

In this study, we have produced nanotextured poly(lactic-co-glycolic acid) (PLGA) films by using polystyrene (PS) particles as a template to make a polydimethylsiloxane mould against which PLGA is solvent cast. Biocompatible, biodegradable and nanotextured PLGA films were prepared with PS particles of diameter of 57, 99, 210, and 280 nm that produced domes of the same dimension in the PLGA surface. The effect of the particulate monolayer templating method was investigated to enable preparation of the films with uniformly ordered surface nanodomes. Cell attachment of a human ovarian cancer cell line (OVCAR3) alone and co-cultured with mesenchymal stem cells (MSCs) was evaluated on flat and topographically nano-patterned surfaces. Cell numbers were observed to increase on the nanotextured surfaces compared to non-textured surfaces both with OVCAR3 cultures and OVCAR3-MSC co-cultures at 24 and 48 h time points

A 3D Heterotypic Breast Cancer Model Demonstrates a Role for Mesenchymal Stem Cells in Driving a Proliferative and Invasive Phenotype

Previous indirect 2D co-culture studies have demonstrated that mesenchymal stem cells (MSCs) promote breast cancer (BC) progression through secretion of paracrine factors including growth factors, cytokines and chemokines. In order to investigate this aspect of the tumour microenvironment in a more relevant 3D co-culture model, spheroids incorporating breast cancer cells (BCCs), both cell lines and primary BCCs expanded as patient-derived xenografts, and MSCs were established. MSCs in co-cultures were shown to enhance proliferation of estrogen receptor (ER)/progesterone receptor (PR)-positive BCCs. In addition, co-culture resulted in downregulation of E-cadherin in parallel with upregulation of the epithelial-mesenchymal transition (EMT)-relation transcription factor, SNAIL. Cytoplasmic relocalization of ski-related novel protein N (SnON), a negative regulator of transforming growth factor-beta (TGF-β) signalling, and of β-catenin, involved in a number of pathways including Wnt signalling, was also observed in BCCs in co-cultures in contrast to monocultures. In addition, the β-catenin inhibitor, 3-[[(4-methylphenyl)sulfonyl]amino]-benzoic acid methyl ester (MSAB), mediated reduced growth and invasion in the co-cultures. This study highlights the potential role for SnON as a biomarker for BC invasiveness, and the importance of interactions between TGF-β and Wnt signalling, involving SnON. Such pathways may contribute towards identifying possible targets for therapeutic intervention in BC patients

Modulation of the tumour promoting functions of cancer associated fibroblasts by phosphodiesterase type 5 inhibition increases the efficacy of chemotherapy in human preclinical models of esophageal adenocarcinoma

Background and aims: Esophageal adenocarcinoma (EAC) is chemoresistant in the majority of cases. The tumor-promoting biology of cancer associated fibroblasts (CAF) make them a target for novel therapies. Phosphodiesterase type 5 inhibitors (PDE5i) have been shown to regulate the activated fibroblast phenotype in benign disease. We investigated the potential for CAF modulation in EAC using PDE5i to enhance the efficacy of chemotherapy. Methods: EAC fibroblasts were treated with PDE5i and phenotypic effects examined using immunoblotting, immunohistochemistry, gel contraction, transwell invasion, organotypics, single cell RNAseq and shotgun proteomics. The combination of PDE5i with standard-of-care chemotherapy (Epirubicin, 5-Fluorouracil and Cisplatin) was tested for safety and efficacy in validated near-patient model systems (3D tumor growth assays (3D-TGAs) and patient derived xenograft (PDX) mouse models). Results: PDE5i treatment reduced alpha-SMA expression in CAFs by 50% (p<0.05), associated with a significant reduction in the ability of CAFs to contract collagen-1 gels and induce cancer cell invasion, (p<0.05). RNAseq and proteomic analysis of CAF and EAC cell lines revealed PDE5i specific regulation of pathways related to fibroblast activation and tumor promotion. 3D-TGA assays confirmed the importance of stromal cells to chemoresistance in EAC, which could be attenuated by PDE5i. Chemotherapy+PDE5i in PDX-bearing mice was safe and significantly reduced PDX tumor volume (p<0.05). Conclusion: PDE5 is a candidate for clinical trials to alter the fibroblast phenotype in esophageal cancer. We demonstrate the specificity of PDE5i for fibroblasts to prevent transdifferentiation and revert the CAF phenotype. Finally, we confirm the efficacy of PDE5i in combination with chemotherapy in close-to-patient in vitro and in vivo PDX-based model systems

Translating genome-wide association findings into new therapeutics for psychiatry

Genome-wide association studies (GWAS) in psychiatry, once they reach sufficient sample size and power, have been enormously successful. The Psychiatric Genomics Consortium (PGC) aims for mega-analyses with sample sizes that will grow to (cumulatively) >1 million individuals in the next 5 years. This should lead to hundreds of new findings for common genetic variants across nine psychiatric disorders studied by the PGC. The new targets discovered by GWAS have the potential to restart largely stalled psychiatric drug development pipelines, and the translation of GWAS findings into the clinic is a key aim of the recently funded phase 3 of the PGC. This is not without considerable technical challenges. These approaches complement the other main aim of GWAS studies on risk prediction approaches for improving detection, differential diagnosis, and clinical trial design. This paper outlines the motivations, technical and analytical issues, and the plans for translating PGC3 findings into new therapeutics

Targeting hypoxia regulated sodium driven bicarbonate transporters reduces triple negative breast cancer metastasis

Regions of low oxygen (hypoxia) are found in >50% of breast tumours, most frequently in the more aggressive triple negative breast cancer subtype (TNBC). Metastasis is the cause of 90% of breast cancer patient deaths. Regions of tumour hypoxia tend to be more acidic and both hypoxia and acidosis increase tumour metastasis. In line with this the metastatic process is dependent on pH regulatory mechanisms. We and others have previously identified increased hypoxic expression of Na+ driven bicarbonate transporters (NDBTs) as a major mechanism of tumour pH regulation. Hypoxia induced the expression of NDBTs in TNBC, most frequently SLC4A4 and SLC4A5. NDBT inhibition (S0859) and shRNA knockdown suppressed migration (40% reduction) and invasion (70% reduction) in vitro. Tumour xenograft metastasis in vivo was significantly reduced by NDBT knockdown. To investigate the mechanism by which NDBTs support metastasis, we investigated their role in regulation of phospho-signalling, epithelial-to-mesenchymal transition (EMT) and metabolism. NDBT knockdown resulted in an attenuation in hypoxic phospho-signalling activation; most notably LYN (Y397) reduced by 75%, and LCK (Y394) by 72%. The metastatic process is associated with EMT. We showed that NDBT knockdown inhibited EMT, modulating the expression of key EMT transcription factors and ablating the expression of vimentin whilst increasing the expression of E-cadherin. NDBT knockdown also altered metabolic activity reducing overall ATP and extracellular lactate levels. These results demonstrate that targeting hypoxia-induced NDBT can be used as an approach to modulate phospho-signalling, EMT, and metabolic activity and reduce tumour migration, invasion, and metastasis in vivo

Application of a 3D hydrogel-based model to replace use of animals for passaging patient-derived xenografts

PurposeThis 3D in vitro cancer model for propagation of patient-derived cells, using a synthetic self-assembling peptide gel, allows the formation of a fully characterised, tailorable tumour microenvironment. Unlike many existing 3D cancer models, the peptide gel is inert, apart from molecules and motifs deliberately added or produced by cells within the model.MethodsBreast cancer patient-derived xenografts (PDXs) were disaggregated and embedded in a peptide hydrogel. Growth was monitored by microscopic examination and at intervals, cells were extracted from the gels and passaged on into fresh gels. Passaged cells were assessed by qPCR and immunostaining techniques for the retention of characteristic markers.ResultsBreast cancer PDXs were shown to be capable of expansion over four or more passages in the peptide gel. Contaminating mouse cells were found to be rapidly removed by successive passages. The resulting human cells were shown to be compatible with a range of common assays useful for assessing survival, growth and maintenance of heterogeneity.ConclusionsBased on these findings, the hydrogel has the potential to provide an effective and practical breast cancer model for the passage of PDXs which will have the added benefits of being relatively cheap, fully-defined and free from the use of animals or animal products. Encapsulated cells will require further validation to confirm the maintenance of cell heterogeneity, genotypes and phenotypes across passage, but with further development, including the addition of bespoke cell and matrix components of the tumour microenvironment, there is clear potential to model other cancer types