Regioregular alternating polyampholytes have enhanced biomimetic ice recrystallization activity compared to random copolymers and the role of side chain verses main chain hydrophobicity

Antifreeze proteins from polar fish species are potent ice recrystallization inhibitors (IRIs) effectively stopping all ice growth. Additives which have IRI activity have been shown to enhance cellular cryopreservation with potential to improve the distribution of donor cells and tissue. Polyampholytes, polymers with both anionic and cationic side chains, are a rapidly emerging class of polymer cryoprotectants, but their mode of action and the structural features essential for activity are not clear. Here regio-regular polyampholytes are synthesized from maleic anhydride co-polymers to enable stoichiometric installation of the charged groups, ensuring regio-regularity which is not possible using conventional random co-polymerisation. A modular synthetic strategy is employed to enable the backbone and side chain hydrophobicity to be varied, with side chain hydrophobicity found to have a profound effect on the IRI activity. The activity of the regio-regular polymers was found to be superior to those derived from a standard random copolymerisation with statistical incorporation of monomers, demonstrating that sequence composition is crucial to the activity of IRI active polyampholytes

Multivalent antimicrobial polymer nanoparticles target mycobacteria and gram-negative bacteria by distinct mechanisms

Due to the emergence of antimicrobial resistance to traditional small molecule drugs, cationic antimicrobial polymers are appealing targets. Mycobacterium tuberculosis is a particular problem, with multi- and total drug resistance spreading and more than a billion latent infections globally. This study reports nanoparticles bearing variable densities of poly(dimethylaminoethyl methacrylate) and the unexpected and distinct mechanisms of action this multivalent presentation imparts against Escherichia coli verses Mycobacterium smegmatis (model of M. tuberculosis), leading to killing or growth inhibition respectively. A convergent ‘grafting to’ synthetic strategy was used to assemble a 50-member nanoparticle library and using a high- throughput screen identified that only the smallest (2 nm) particles were stable in both saline and complex cell media. Compared to the linear polymers, the nanoparticles displayed 2- and 8-fold enhancements in antimicrobial activity against M. smegmatis and E. coli respectively. Mechanistic studies demonstrated that the antimicrobial particles were bactericidal against E. coli, due to rapid disruption of the cell membranes. Conversely, against M. smegmatis the particles did not lyse the cell membrane but rather had a bacteriostatic effect. These results demonstrate that to develop new polymeric anti-tuberculars the widely assumed, broad spectrum, membrane-disrupting mechanism of polycations must be re-evaluated. It is clear that synthetic nanomaterials can engage in more complex interactions with mycobacteria, which we hypothesise is due to the unique cell envelope at the surface of these bacteria

Recurrent pregnancy loss is associated with a pro-senescent decidual response during the peri-implantation window

During the implantation window, the endometrium becomes poised to transition to a pregnant state, a process driven by differentiation of stromal cells into decidual cells (DC). Perturbations in this process, termed decidualization, leads to breakdown of the feto-maternal interface and miscarriage, but the underlying mechanisms are poorly understood. Here, we reconstructed the decidual pathway at single-cell level in vitro and demonstrate that stromal cells first mount an acute stress response before emerging as DC or senescent DC (snDC). In the absence of immune cell-mediated clearance of snDC, secondary senescence transforms DC into progesterone-resistant cells that abundantly express extracellular matrix remodelling factors. Additional single-cell analysis of midluteal endometrium identified DIO2 and SCARA5 as marker genes of a diverging decidual response in vivo. Finally, we report a conspicuous link between a pro-senescent decidual response in peri-implantation endometrium and recurrent pregnancy loss, suggesting that pre-pregnancy screening and intervention may reduce the burden of miscarriage

Epigenetic priming of immune/inflammatory pathways activation and abnormal activity of cell cycle pathway in a perinatal model of white matter injury

Prenatal inflammatory insults accompany prematurity and provoke diffuse white matter injury (DWMI), which is associated with increased risk of neurodevelopmental pathologies, including autism spectrum disorders. DWMI results from maturation arrest of oligodendrocyte precursor cells (OPCs), a process that is poorly understood. Here, by using a validated mouse model of OPC maturation blockade, we provide the genome-wide ID card of the effects of neuroinflammation on OPCs that reveals the architecture of global cell fate issues underlining their maturation blockade. First, we find that, in OPCs, neuroinflammation takes advantage of a primed epigenomic landscape and induces abnormal overexpression of genes of the immune/inflammatory pathways: these genes strikingly exhibit accessible chromatin conformation in uninflamed OPCs, which correlates with their developmental, stage-dependent expression, along their normal maturation trajectory, as well as their abnormal upregulation upon neuroinflammation. Consistently, we observe the positioning on DNA of key transcription factors of the immune/inflammatory pathways (IRFs, NFkB), in both unstressed and inflamed OPCs. Second, we show that, in addition to the general perturbation of the myelination program, neuroinflammation counteracts the physiological downregulation of the cell cycle pathway in maturing OPCs. Neuroinflammation therefore perturbs cell identity in maturing OPCs, in a global manner. Moreover, based on our unraveling of the activity of genes of the immune/inflammatory pathways in prenatal uninflamed OPCs, the mere suppression of these proinflammatory mediators, as currently proposed in the field, may not be considered as a valid neurotherapeutic strategy

Computational approaches for determination of transcriptomic and epigenomic states

Over the last half century, major advances in molecular biology methods and technologies have inspired significant progress in research across all branches of life sciences. In particular, transcriptomic and epigenomic profiling have allowed for detailed studies of gene regulation, as data from both techniques reflect a multitude of biological processes and responses. As such, there exists a need for computational approaches that can recognise the associated biological states with confidence, which is the main focus of this thesis. First, chapter 3 presents a novel tool - JACC (Julia’s Algorithm for Cell Classification) the Ripper - for cell clustering of scRNA-Seq data, which takes advantage of the intrinsic multimodality of gene expression profiles within such datasets to split the cells into their corresponding cell types. This tool is among the first of its kind, offering multiple advantages over standard clustering work-flows, including its ability to identify rare cell populations and generation of a report clearly documenting the reasons behind each splitting decision thereby providing evidence and transparency to the user. As such, JACC can be used in any scRNA-Seq research setting. Second, chapter 4 introduces a novel method - ESPAM (Endometrial Secretory Phase Assignment Model) - for the assessment of secretory phase progress of endometrial biopsy samples based on a specifically designed RT-qPCR gene expression measurement assay. The model combines data binning with probability density estimates to obtain timing predictions of samples, alongside also providing a measure for tissue asynchrony which flags up any samples with unusually noisy gene measurements. ESPAM can be used in both clinical and research environments, as the model can facilitate any endometrial analyses of the secretory phase, which could improve our understanding of reproductive disorders and inform decisions with regards to any required medical treatments or lack thereof. Third, chapter 5 describes a comparative approach, which showed that differentially open chromatin regions of bulk ATAC-Seq data can be used as a signature for the recognition of global chromatin states. Moreover, such comparisons can be effective even across species, providing for a valuable method that can be used to extend standard chromatin accessibility analyses. As such, this method could be used in any scenario that aims to compare chromatin states arising from similar treatment conditions across two or more datasets, possibly even for more distant species

Regioregular Alternating Polyampholytes Have Enhanced Biomimetic Ice Recrystallization Activity Compared to Random Copolymers and the Role of Side Chain versus Main Chain Hydrophobicity

Antifreeze proteins from polar fish species are potent ice recrystallization inhibitors (IRIs) effectively stopping all ice growth. Additives that have IRI activity have been shown to enhance cellular cryopreservation with potential to improve the distribution of donor cells and tissue. Polyampholytes, polymers with both anionic and cationic side chains, are a rapidly emerging class of polymer cryoprotectants, but their mode of action and the structural features essential for activity are not clear. Here regioregular polyampholytes are synthesized from maleic anhydride copolymers to enable stoichiometric installation of the charged groups, ensuring regioregularity, which is not possible using conventional random copolymerization. A modular synthetic strategy is employed to enable the backbone and side chain hydrophobicity to be varied, with side chain hydrophobicity found to have a profound effect on the IRI activity. The activity of the regioregular polymers was found to be superior to those derived from a standard random copolymerization with statistical incorporation of monomers, demonstrating that sequence composition is crucial to the activity of IRI active polyampholytes

EndoTime : non-categorical timing estimates for luteal endometrium

STUDY QUESTION Can the accuracy of timing of luteal phase endometrial biopsies based on urinary ovulation testing be improved by measuring the expression of a small number of genes and a continuous, non-categorical modelling approach

Epigenome and transcriptome landscapes highlight dual roles of proinflammatory players in a perinatal model of white matter injury

Premature birth is the commonest cause of death and disability in young children. Diffuse white matter injury (DWMI), provoked by inflammatory insults accompanying prematurity, is associated with increased risk of neurodevelopmental disorders – such as autism spectrum disorders – and is due to maturation arrest in oligodendrocyte precursors (OPCs). The lack of therapeutic solutions is a strong impetus to unveil the molecular mechanisms underlying neuroinflammation impact on OPC cell fate. We used a validated mouse model of DWMI, induced by systemic- and neuro-inflammation – as observed in preterm infants – and based on interleukin-1B administration from postnatal day P1 to P5. Using integrated genome-wide approaches we showed that neuroinflammation induced limited epigenomic disturbances in OPCs, but marked transcriptomic alterations of genes of the immune/inflammatory pathways. We found that these genes were expressed in control OPCs and physiologically downregulated between P3-P10, as part of the OPC normal developmental trajectory. We observed that transcription factors of the inflammatory pathways occupied DNA both in unstressed and inflamed OPCs. Thus, rather than altering genome-wide chromatin accessibility, neuroinflammation takes advantage of open chromatin regions and deeply counteracts the stage-dependent downregulation of these active transcriptional programs by the sustained upregulation of transcript levels. The intricate dual roles – stress-responsive and potentially developmental – of these proinflammatory mediators strongly suggest that the mere suppression of these inflammatory mediators, as currently proposed, may not be a valid neurotherapeutic strategy. Our study provides new insights for the future development of more targeted approaches to protect the preterm brain