Biology subjects

Philips, A.

Publications and source records attributed to Philips, A..

10 recordsLinked to original sources

bioRxiv2025

Whole-miRNome sequencing (WMS) - a panel for targeted sequencing of all human miRNA genes

There is a growing interest in the genetic variation of noncoding genomic elements, including miRNAs, and several mutations in miRNA genes implicated in human diseases, including cancer, have already been detected. However, the lack of dedicated analytical tools severely hampers progress in this area. In this study, we developed whole-miRNome sequencing (WMS), which enables targeted sequencing of all human miRNA genes (n[~]2000) and 28 miRNA biogenesis genes. Herein, by sequencing almost 600 samples, including [~]300 tumor/normal pairs of samples from different cancer types, we identified [~]2, 000 mutations, including 1, 435 cancer somatic mutations, with 879 occurring in miRNA genes. These mutations were located in all parts of the genes, including seed or cleavage sites essential for the functioning of miRNA genes. The high reliability of the mutations was confirmed through various approaches, including different sequencing methods. The analysis identified several miRNA genes with functional enrichment of cancer mutations, including MIR3928, specifically mutated in basal cell carcinoma (BCC), indicating its potential role in this cancer. WMS also allowed the identification of multiple copy number alterations, hotspots of which often encompassed miRNA genes. WMS provides highly effective, low-cost sequencing of all miRNA genes in different types of samples, including highly degraded FFPE samples.

genomics↗

bioRxiv2024

Conserved heavy/light contacts and germline preferences revealed by a large-scale analysis of natively paired human antibody sequences and structural data.

Antibody next-generation sequencing (NGS) datasets have become crucial to develop computational models addressing this successful class of therapeutics. Although antibodies are composed of both heavy and light chains, most NGS sequencing depositions provide them in unpaired form, reducing their utility. Here we introduce PairedAbNGS, a novel database with paired heavy/light antibody chains. To the best of our knowledge, this is the largest resource for paired natural antibody sequences with 58 bioprojects and over 14 million assembled productive sequences. We make the database accessible at http://naturalantibody.com/paired-ngs as a valuable tool for biological and machine-learning applications. Using this dataset, we investigated heavy and light chain variable (V) gene pairing preferences and found significant biases beyond gene usage frequencies, possibly due to receptor editing favoring less autoreactive combinations. Analyzing the available antibody structures from the Protein Data Bank, we studied conserved contact residues between heavy and light chains, particularly interactions between the CDR3 region of one chain and the FWR2 region of the opposite chain. Examination of amino acid pairs at key contact sites revealed significant deviations of amino acids distributions compared to random pairings, in the heavy chains CDR3 region contacting the opposite chain, indicating specific interactions might be crucial for proper chain pairing. This observation is further reinforced by preferential IGHV-IGLJ and IGLV-IGHJ pairing preferences. We hope that both our resources and the findings would contribute to improving the engineering of biological drugs.

immunology↗

bioRxiv2024

Optimising the production of dsRNA biocontrols in microbial systems using multiple transcriptional terminators.

Crop pests and pathogens annually cause over $100 billion in global crop damage, with insects consuming 5-20% of major grain crops. Current crop pest and disease control strategies rely on insecticidal and fungicidal sprays, plant genetic resistance, transgenes and agricultural practices. dsRNA is emerging as a novel sustainable method of plant protection as an alternative to traditional chemical pesticides. Successful commercialisation of dsRNA based biocontrols requires the economical production of large quantities of dsRNA combined with suitable delivery methods to ensure RNAi efficacy against the target pest. In this study, we have optimised the design of plasmid DNA constructs to produce dsRNA biocontrols in E. coli, by employing a wide range of alternative synthetic transcriptional terminators prior to measurement of dsRNA yield. We demonstrate that a 7.8-fold increase of dsRNA was achieved using triple synthetic transcriptional terminators within a dual T7 dsRNA production system compared to the absence of transcriptional terminators. Moreover, our data demonstrates that batch fermentation production dsRNA using multiple transcriptional terminators is scalable and generates significantly higher yields of dsRNA generated in the absence of transcriptional terminators at both small-scale batch culture and large-scale fermentation. In addition, we show that application of these dsRNA biocontrols expressed in E. coli cells results in increased insect mortality. Finally, novel mass spectrometry analysis was performed to determine the precise sites of transcriptional termination at the different transcriptional terminators providing important further mechanistic insight.

synthetic biology↗

bioRxiv2022

Precise control of neural activity using temporally dithered and spatially multiplexed electrical stimulation

Neural implants have the potential to restore lost sensory function by electrically evoking the complex naturalistic activity patterns of neural populations. However, it can be difficult to predict and control evoked neural responses to simultaneous multi-electrode stimulation due to nonlinearity of the responses. We present a solution to this problem and demonstrate its utility in the context of a bi-directional retinal implant for restoring vision. A dynamically optimized stimulation approach encodes incoming visual stimuli into a rapid, greedily chosen, temporally dithered and spatially multiplexed sequence of simple stimulation patterns. Stimuli are selected to optimize the reconstruction of the visual stimulus from the evoked responses. Temporal dithering exploits the slow time scales of downstream neural processing, and spatial multiplexing exploits the independence of responses generated by distant electrodes. The approach was evaluated using an experimental laboratory prototype of a retinal implant: large-scale, high-resolution multi-electrode stimulation and recording of macaque and rat retinal ganglion cells ex vivo. The dynamically optimized stimulation approach substantially enhanced performance compared to existing approaches based on static mapping between visual stimulus intensity and current amplitude. The modular framework enabled parallel extensions to naturalistic viewing conditions, incorporation of perceptual similarity measures, and efficient implementation for an implantable device. A direct closed-loop test of the approach supported its potential use in vision restoration. Impact StatementA novel method of electrical stimulation to precisely control neural activity for sensory restoration exhibits improvements in visual stimulus reconstruction, enables efficient hardware design, and extends to naturalistic conditions.

neuroscience↗

bioRxiv2022

Translocator protein is a marker of activated microglia in rodent models but not human neurodegenerative diseases

Microglial activation plays central roles in neuro-inflammatory and neurodegenerative diseases. Positron emission tomography (PET) targeting 18kDa Translocator Protein (TSPO) is widely used for localising inflammation in vivo, but its quantitative interpretation remains uncertain. We show that TSPO expression increases in activated microglia in mouse brain disease models but does not change in a non-human primate disease model or in common neurodegenerative and neuroinflammatory human diseases. We describe genetic divergence in the TSPO gene promoter, consistent with the hypothesis that the increase in TSPO expression in activated myeloid cells is unique to a subset of species within the Muroidea superfamily of rodents. We show that TSPO is mechanistically linked to classical pro-inflammatory myeloid cell function in rodents but not humans. These data emphasise that TSPO expression in human myeloid cells is related to different phenomena than in mice, and that TSPO PET reflects density of inflammatory cells rather than activation state.

neuroscience↗

bioRxiv2022

CLN7 mutation causes aberrant redistribution of protein isoforms and contributes to Batten disease pathobiology

The variant late infantile form of the inherited neurodegenerative Batten disease (BD) is caused by mutations in the CLN7/MFSD8 gene and represents a strong candidate for gene therapy. Post-natal intracerebral administration of AAV9-hCLN7 to Cln7{Delta}ex2 knockout mice resulted in extended lifespan but dose escalation resulted in reduced acuity in neurophysiology tests, cerebral atrophy and elevated neuroinflammation. Comparing patient and control iPSC-derived neural progenitor cells (iNPC) we discovered that CLN7 localizes to the nucleus as well as the endolysosomal network and is differentially distributed in BD iNPC. Proteomics identified a profound nuclear defect in BD iNPC that compounds with mitochondrial and lysosomal metabolic defects resulting in elevated apoptosis. We further identified a 50kDa common nuclear CLN7 isoform and a 37kDa isoform that accumulates only in BD iNPC nuclei. Our findings suggest that successful treatment of CLN7 BD will require combinatorial therapies addressing both loss and aberrant gain of protein function.

cell biology↗

bioRxiv2022

Chemically modified dsRNA induces RNAi effects in insects in vitro and in vivo: A potential new tool for improving RNA-based plant protection

Global agriculture loses over $100 billion of produce annually to crop pests such as insects. Many of these crop pests either have no current means of control or have developed resistance against chemical pesticides. Long dsRNAs are capable of inducing RNA interference (RNAi) in insects and are emerging as novel highly selective alternatives for sustainable insect management strategies. However, there are significant challenges associated with RNAi efficacy in insects. In this study, we synthesised a range of chemically modified long dsRNA in an approach to improve nuclease resistance and RNAi efficacy in insects. The results showed that dsRNA containing phosphorothioate modifications demonstrated increased resistance to southern green stink bug saliva nucleases. Phosphorothioate and 2-fluoro modified dsRNA also demonstrated increased resistance to degradation by soil nucleases and increased RNAi efficacy in Drosophila melanogaster cell cultures. In live insects, chemically modified long dsRNA successfully resulted in mortality in both stink bug and corn rootworm. The results provide further mechanistic insight of RNAi efficacy dependence on modifications in the sense or antisense strand of the dsRNA in insects and demonstrate for the first time that RNAi can successfully be triggered by chemically modified long dsRNA in insect cells or live insects.

bioengineering↗

bioRxiv2021

Profile of basal cell carcinoma mutations and copy number alterations - focus on gene-associated noncoding variants

Basal cell carcinoma (BCC) of the skin is the most common cancer in humans, characterized by the highest mutation rate among cancers, and is mostly driven by mutations in genes involved in the hedgehog pathway. To date, almost all BCC genetic studies have focused exclusively on protein-coding sequences; therefore, the impact of noncoding variants on the BCC genome is unrecognized. In this study, with the use of whole-exome sequencing of 27 tumor/normal pairs of BCC samples, we performed an analysis of somatic mutations in both protein-coding sequences and gene-associated noncoding regions, including 5UTRs, 3UTRs, and exon-adjacent intron sequences. Separately, in each region, we performed hotspot identification, mutation enrichment analysis, and cancer driver identification with OncodriveFML. Additionally, we performed a whole-genome copy number alteration analysis with GISTIC2. Of the >80,000 identified mutations, [~]50% were localized in noncoding regions. The results of the analysis generally corroborated the previous findings regarding genes mutated in coding sequences, including PTCH1, TP53, and MYCN, but more importantly showed that mutations were also clustered in specific noncoding regions, including hotspots. Some of the genes specifically mutated in noncoding regions were identified as highly potent cancer drivers, of which BAD had a mutation hotspot in the 3UTR, DHODH had a mutation hotspot in the Kozak sequence in the 5UTR, and CHCHD2 frequently showed mutations in the 5UTR. All of these genes are functionally implicated in cancer-related processes (e.g., apoptosis, mitochondrial metabolism, and de novo pyrimidine synthesis) or the pathogenesis of UV radiation-induced cancers. We also found that the identified BAD and CHCHD2 mutations frequently occur in melanoma but not in other cancers via The Cancer Genome Atlas analysis. Finally, we identified frequent deletion of chr9q, encompassing PTCH1, and unreported frequent copy number gain of chr9p, encompassing the genes encoding the immune checkpoint ligands PD-L1 and PD-L2. In conclusion, this study is the first systematic analysis of coding and noncoding mutations in BCC and provides a strong basis for further analyses of the variants in BCC and cancer in general. Author summaryThe study is the first systematic analysis of both coding and noncoding mutations in basal cell carcinoma (BCC), the most common and the most highly mutated human cancer. Noncoding mutations accounted for [~]50% ([~]40K mutations) of all mutations detected by the standard WES approach. Among the genes frequently mutated in noncoding regions are: BAD with a hotspot in the 3UTR, DHODH with a hotspot in the Kozak sequence, and CHCHD2 with mutations in 5UTR, all genes functionally related to cell metabolism and apoptosis. Analysis of copy number alterations showed frequent chr9q deletion, encompassing PTCH1 (the key BCC tumor suppressor), and frequent copy number gain of chr9p, encompassing the genes of the immune checkpoint proteins PD-L1 and PD-L2.

cancer biology↗

bioRxiv2021

Experimental Confirmation of Multiple Co-Existent DNA Secondary Structures using Low-Yield Bisulfite Sequencing

Predicting DNA secondary structures is critical to a broad range of applications involving single-stranded DNA (ssDNA), yet remains an open problem. Existing prediction models are limited by insufficient experimental data, due to a lack of high-throughput methods to study DNA structures, in contrast to RNA structures. Here, we present a method for profiling DNA secondary structures using multiplexed low-yield bisulfite sequencing (MLB-seq), which examines the chemical accessibility of cytosines in thousands of different oligonucleotides. By establishing a probability-based model to evaluate the consensus probability between MLB-seq data and structures proposed using NUPACK software, we identified the secondary structures of individual ssDNA molecules and estimated the distribution of multiple secondary structures in solution. We studied the structures of 1,057 human genome subsequences and experimentally confirmed that 84% adopted two or more structures. MLB-seq thus enables high-throughput ssDNA structure profiling and will benefit the design of probes, primers, aptamers, and genetic regulators.

molecular biology↗

bioRxiv2018

Global increase in circRNA levels in myotonic dystrophy

Splicing aberrations induced as a consequence of the sequestration of MBNL splicing factors on the DMPK transcript, which contains expanded CUG repeats, present a major pathomechanism of myotonic dystrophy type 1 (DM1). As MBNLs may also be important factors involved in the biogenesis of circular RNAs (circRNAs), we hypothesized that the level of circRNAs would be decreased in DM1. To test this hypothesis, we selected twenty well-validated circRNAs and analyzed their levels in several experimental systems (e.g., cell lines, DM muscle tissues, and a mouse model of DM1) using droplet digital PCR assays. We also explored the global level of circRNAs using two RNA-Seq datasets of DM1 muscle samples. Contrary to our original hypothesis, our results consistently showed a global increase in circRNA levels in DM1 and we identified numerous circRNAs that were increased in DM1. We also identified many genes (including muscle-specific genes) giving rise to numerous (>10) circRNAs. Thus, this study is the first to show an increase in global circRNA levels in DM1. We also provided preliminary results showing the association of circRNA level with muscle weakness and alternative splicing changes that are biomarkers of DM1 severity. Author SummaryRecently, a great deal of interest has been focused on a new class of RNA molecules called circular RNAs (circRNAs). To date, thousands of circRNAs have been found in different human cells/tissues. Although the function of circRNAs remains mostly unknown, circRNAs have emerged as an important component of the RNA-RNA and RNA-protein interactome. Thus, intensive efforts are being made to fully understand the biology and function of circRNAs, especially their role in human diseases. As an important role in the biogenesis of circRNA may be played by MBNL splicing factors, in this study we used DM1 (to a lesser extent, DM2) as a natural model in which the level of MBNLs is decreased. In contrast to the expected effect, our results consistently showed a global increase in circRNA levels in DM1. As a consequence, whole genome transcriptome analysis revealed dozens of circRNAs with significantly altered (mostly increased) levels in DM1. Furthermore, we observed that the circRNA levels were in many cases strongly associated with DM1 severity.

molecular biology↗