Biology subjects

Smith, C.

Publications and source records attributed to Smith, C..

At least 109 records · Page 6Linked to original sources

bioRxiv2023

The Genomic Legacy of Human Management and sex-biased Aurochs hybridization in Iberian Cattle

Cattle have been a valuable economic resource and cultural icon since prehistory. From the initial expansion of domestic cattle into Europe during the Neolithic period, taurine cattle (Bos taurus) and their wild ancestor, the aurochs (B. primigenius), had overlapping ranges, leading to ample opportunities for mating (whether intended by farmers or not). We performed a bioarchaeological analysis of 24 Bos remains from Iberia dating from the Mesolithic to the Roman period. The archaeogenomic dataset allows us to investigate the extent of domestic-wild hybridization over time, providing insight into the species behavior and human hunting and management practices by aligning changes with cultural and genomic transitions in the archaeological record. Our results show frequent hybridization during the Neolithic and Chalcolithic, likely reflecting a mix of hunting and herding or relatively unmanaged herds, with mostly male aurochs and female domestic cattle involved in hybridization. This is supported by isotopic evidence consistent with ecological niche sharing, with only a few domestic cattle possibly being managed. The proportion of aurochs ancestry remains relatively constant from about 4000 years ago, probably due to herd management and selection against first generation hybrids, coinciding with other cultural transitions. The constant level of wild ancestry ({bsim}20%) continues into modern western European breeds including the Spanish Lidia breed which is bred for its aggressiveness and fighting ability, but does not display elevated levels of aurochs ancestry. This study takes a genomic glance at the impact of human actions and wild introgression in the establishment of cattle as one of the most important domestic species today.

genomics↗

bioRxiv2023

A multistage Plasmodium CRL4-WIG1 ubiquitin ligase is critical for the formation of functional microtubule organisation centres in microgametocytes

Malaria is a mosquito-borne infectious disease caused by unicellular eukaryotic parasites of the Plasmodium genus. Protein ubiquitination by E3 ligases is a critical post-translational modification required for various cellular processes during the lifecycle of Plasmodium parasites. However, little is known about the repertoire and function of these enzymes in Plasmodium. Here we show that Plasmodium expresses a conserved cullin RING E3 ligase (CRL) complex that is functionally related to the eukaryotic CRL4. In P. falciparum asexual blood stages, a cullin-4 scaffold interacts with the RING protein RBX1, the adaptor protein DDB1 and a set of putative receptor proteins that may determine substrate specificity for ubiquitination. These receptor proteins contain WD40-repeat domains and include WD-repeat protein Important for Gametogenesis 1 (WIG1). This CRL4-related complex is also expressed in P. berghei gametocytes, with WIG1 being the only putative receptor detected in both schizont and gametocyte stages. While WIG1 is not required for the proliferation of P. berghei asexual blood stages, its disruption leads to a complete block in microgamete formation. Proteomic analyses indicate that WIG1 disruption alters proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. Further analysis by ultrastructure expansion microscopy (U-ExM) indicates that WIG1-dependent depletion of ciliary proteins is associated with impaired formation of the microtubule organisation centres that coordinate mitosis with axoneme formation and altered DNA replication during microgametogenesis. This work identifies a CRL4-related ubiquitin ligase in Plasmodium that is critical for the transmission of malaria parasites by regulating proteostasis of ciliary and DNA replication proteins.

microbiology↗

bioRxiv2023

Dichotomy of neutralizing antibody, B cell and T cell responses to SARS-CoV-2 vaccination and protection in healthy adults

Heterogeneity in SARS-CoV-2 vaccine responses is not understood. Here, we identify four patterns of live-virus neutralizing antibody responses: individuals with hybrid immunity (with confirmed prior infection); rare individuals with low responses (paucity of S1-binding antibodies); and surprisingly, two further groups with distinct serological repertoires. One group - broad responders - neutralize a range of SARS-CoV-2 variants, whereas the other - narrow responders - neutralize fewer, less divergent variants. This heterogeneity does not correlate with Ancestral S1-binding antibody, rather the quality of the serological response. Furthermore, IgDlowCD27-CD137+ B cells and CCR6+ CD4+ T cells are enriched in broad responders before dose 3. Notably, broad responders have significantly longer infection-free time after their third dose. Understanding the control and persistence of these serological profiles could allow personalized approaches to enhance serological breadth after vaccination.

immunology↗

bioRxiv2023

A multiadjuvant polysaccharide-amino acid-lipid (PAL) subunit nanovaccine generates robust systemic and lung-specific mucosal immune responses against SARS-CoV-2 in mice

Existing parenteral SARS-CoV-2 vaccines produce only limited mucosal responses, which are essential for reducing transmission and achieving sterilizing immunity. Appropriately designed mucosal boosters could overcome the shortcomings of parenteral vaccines and enhance pre- existing systemic immunity. Here we present a new protein subunit nanovaccine using multiadjuvanted (e.g. RIG-I: PUUC, TLR9: CpG) polysaccharide-amino acid-lipid nanoparticles (PAL-NPs) that can be delivered both intramuscularly (IM) and intranasally (IN) to generate balanced mucosal-systemic SARS-CoV-2 immunity. Mice receiving IM-Prime PUUC+CpG PAL- NPs, followed by an IN-Boost, developed high levels of IgA, IgG, and cellular immunity in the lung, and showed robust systemic humoral immunity. Interestingly, as a purely intranasal vaccine (IN-Prime/IN-Boost), PUUC+CpG PAL-NPs induced stronger lung-specific T cell immunity than IM-Prime/IN-Boost, and a comparable IgA and neutralizing antibodies, although with a lower systemic antibody response, indicating that a fully mucosal delivery route for SARS-CoV-2 vaccination may also be feasible. Our data suggest that PUUC+CpG PAL-NP subunit vaccine is a promising candidate for generating SARS-CoV-2 specific mucosal immunity.

bioengineering↗

bioRxiv2023

Benchmarking splice variant prediction algorithms using massively parallel splicing assays

BackgroundVariants that disrupt mRNA splicing account for a sizable fraction of the pathogenic burden in many genetic disorders, but identifying splice-disruptive variants (SDVs) beyond the essential splice site dinucleotides remains difficult. Computational predictors are often discordant, compounding the challenge of variant interpretation. Because they are primarily validated using clinical variant sets heavily biased to known canonical splice site mutations, it remains unclear how well their performance generalizes. ResultsWe benchmarked eight widely used splicing effect prediction algorithms, leveraging massively parallel splicing assays (MPSAs) as a source of experimentally determined ground-truth. MPSAs simultaneously assay many variants to nominate candidate SDVs. We compared experimentally measured splicing outcomes with bioinformatic predictions for 3,616 variants in five genes. Algorithms concordance with MPSA measurements, and with each other, was lower for exonic than intronic variants, underscoring the difficulty of identifying missense or synonymous SDVs. Deep learning-based predictors trained on gene model annotations achieved the best overall performance at distinguishing disruptive and neutral variants. Controlling for overall call rate genome-wide, SpliceAI and Pangolin also showed superior overall sensitivity for identifying SDVs. Finally, our results highlight two practical considerations when scoring variants genome-wide: finding an optimal score cutoff, and the substantial variability introduced by differences in gene model annotation, and we suggest strategies for optimal splice effect prediction in the face of these issues. ConclusionSpliceAI and Pangolin showed the best overall performance among predictors tested, however, improvements in splice effect prediction are still needed especially within exons.

bioinformatics↗

bioRxiv2023

Melanoma clonal subline analysis uncovers heterogeneity-driven immunotherapy resistance mechanisms

Intratumoral heterogeneity (ITH) can promote cancer progression and treatment failure, but the complexity of the regulatory programs and contextual factors involved complicates its study. To understand the specific contribution of ITH to immune checkpoint blockade (ICB) response, we generated single cell-derived clonal sublines from an ICB-sensitive and genetically and phenotypically heterogeneous mouse melanoma model, M4. Genomic and single cell transcriptomic analyses uncovered the diversity of the sublines and evidenced their plasticity. Moreover, a wide range of tumor growth kinetics were observed in vivo, in part associated with mutational profiles and dependent on T cell-response. Further inquiry into melanoma differentiation states and tumor microenvironment (TME) subtypes of untreated tumors from the clonal sublines demonstrated correlations between highly inflamed and differentiated phenotypes with the response to anti-CTLA-4 treatment. Our results demonstrate that M4 sublines generate intratumoral heterogeneity at both levels of intrinsic differentiation status and extrinsic TME profiles, thereby impacting tumor evolution during therapeutic treatment. These clonal sublines proved to be a valuable resource to study the complex determinants of response to ICB, and specifically the role of melanoma plasticity in immune evasion mechanisms.

cancer biology↗

bioRxiv2023

The small mycobacterial ribosomal protein, bS22, modulates aminoglycoside accessibility to its 16S rRNA helix-44 binding site

Treatment of tuberculosis continues to be challenging due to the widespread latent form of the disease and the emergence of antibiotic-resistant strains of the pathogen, Mycobacterium tuberculosis. Bacterial ribosomes are a common and effective target for antibiotics. Several second line anti-tuberculosis drugs, e.g. kanamycin, amikacin, and capreomycin, target ribosomal RNA to inhibit protein synthesis. However, M. tuberculosis can acquire resistance to these drugs, emphasizing the need to identify new drug targets. Previous cryo-EM structures of the M. tuberculosis and M. smegmatis ribosomes identified two novel ribosomal proteins, bS22 and bL37, in the vicinity of two crucial drug-binding sites: the mRNA-decoding center on the small (30S), and the peptidyl-transferase center on the large (50S) ribosomal subunits, respectively. The functional significance of these two small proteins is unknown. In this study, we observe that an M. smegmatis strain lacking the bs22 gene shows enhanced susceptibility to kanamycin compared to the wild-type strain. Cryo-EM structures of the ribosomes lacking bS22 in the presence and absence of kanamycin suggest a direct role of bS22 in modulating the 16S rRNA kanamycin-binding site. Our structures suggest that amino-acid residue Lys-16 of bS22 interacts directly with the phosphate backbone of helix 44 of 16S rRNA to influence the micro-configuration of the kanamycin-binding pocket. Our analysis shows that similar interactions occur between eukaryotic homologues of bS22, and their corresponding rRNAs, pointing to a common mechanism of aminoglycoside resistance in higher organisms.

biochemistry↗

bioRxiv2023

The remarkable complexity of the brain microbiome in health and disease

Microbes in human brain and their potential contribution to neurodegenerative conditions such as Alzheimers disease (AD) have long been debated. We recently developed a new method (the electronic tree of life, eToL) based on small subunit ribosomal RNA (rRNA) probes, further confirmed by large subunit rRNA analysis, to comprehensively address the spectrum of microorganisms in control and AD brain. We report a remarkable diversity of brain microbes in control brain. The most abundant are fungi, bacteria, and chloroplastida, and we report detailed identification of representative microbial species. The pattern is substantially conserved across different bilateran species from Drosophila to human. In terms of diversity, the human brain microbiome appears to be a subset (~20%) of the gut microbiome. Adenovirus type C was the major virus found in human brain; other viruses were not well represented. However, the spectrum of brain microbes differed between individuals as well as between brain regions examined from single individuals (amygdala, cingulate cortex, hippocampus, hypothalamus); of these four regions, the highest microbial burden was in cingulate cortex. There was evidence of spreading of pathogens between brain regions in single individuals. Some microbes are over-represented in AD brain according to two measures: (i) absolute number of microbes normalized to endogenous human transcripts, and (ii) the number of brain specimens showing overabundance versus control. Species over-represented in AD brain according to both measures notably include bacteria (Streptococcus, Staphylococcus/Bacillus, Sphingomonas/Ralstonia) and fungi (Acrocalymma/Altenaria/Aureobasidium of the Aspergillus group; Komagataella of the Candida group, Cortinarius of the Schizophyllum group, and Tausonia of the Cryptococcus group), that are all related to known human pathogens. In addition, an uncharacterized chloroplastida (algae-related) species was more abundant in AD brain samples. Although these findings point to diverse microbial species, indicative of multiple causation, similar absolute levels of bacteria and fungi in AD brain samples could suggest synergy between pathogens. However, it is important to stress that not all AD samples were positive for these microbes, but this could be because the affected brain region(s) was not examined. These findings support the contention that infection, perhaps associated with declining immunity with age, may contribute to AD development.

neuroscience↗

bioRxiv2023

Genome-wide mapping of the Escherichia coli PhoB regulon reveals many transcriptionally inert, intragenic binding sites

Genome-scale analyses have revealed many transcription factor binding sites within, rather than upstream of genes, raising questions as to the function of these binding sites. Here, we use complementary approaches to map the regulon of the Escherichia coli transcription factor PhoB, a response regulator that controls transcription of genes involved in phosphate homeostasis. Strikingly, the majority of PhoB binding sites are located within genes, but these intragenic sites are not associated with detectable transcription regulation and are not evolutionarily conserved. Many intragenic PhoB sites are located in regions bound by H-NS, likely due to shared sequence preferences of PhoB and H-NS. However, these PhoB binding sites are not associated with transcription regulation even in the absence of H-NS. We propose that for many transcription factors, including PhoB, binding sites not associated with promoter sequences are transcriptionally inert, and hence are tolerated as genomic "noise". IMPORTANCERecent studies have revealed large numbers of transcription factor binding sites within the genes of bacteria. The function, if any, of the vast majority of these binding sites has not been investigated. Here, we map the binding of the transcription factor PhoB across the Escherichia coli genome, revealing that the majority of PhoB binding sites are within genes. We show that PhoB binding sites within genes are not associated with regulation of the overlapping genes. Indeed, our data suggest that bacteria tolerate the presence of large numbers of non-regulatory, intragenic binding sites for transcription factors, and that these binding sites are not under selective pressure.

microbiology↗

bioRxiv2023

Organoid generation from mouse mammary tumors captures the genetic heterogeneity of clinically relevant copy number alterations

Breast cancer metastases exhibit many different genetic alterations, including copy number amplifications. Using publicly available datasets, we identify copy number amplifications in metastatic breast tumor samples and using our organoid-based metastasis assays, and we validate FGFR1 is amplified in collectively migrating organoids. Because the heterogeneity of breast tumors is increasingly becoming relevant to clinical practice, we demonstrate our organoid method captures genetic heterogeneity of individual tumors.

cancer biology↗

bioRxiv2023

Actin nucleators safeguard replication forks by limiting nascent strand degradation

Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress (RS) and protect replication forks. These responses rely on the formation of Replication Protein A (RPA)-single stranded (ss) DNA complexes, yet this process remains largely uncharacterized. Here we establish that actin nucleation-promoting factors (NPFs) associate with replication forks, promote efficient DNA replication and facilitate association of RPA with ssDNA at sites of RS. Accordingly, their loss leads to deprotection of ssDNA at perturbed forks, impaired ATR activation, global replication defects and fork collapse. Supplying an excess of RPA restores RPA foci formation and fork protection, suggesting a chaperoning role for actin nucleators (ANs) (i.e., Arp2/3, DIAPH1) and NPFs (i.e, WASp, N-WASp) in regulating RPA availability upon RS. We also discover that {beta}-actin interacts with RPA directly in vitro, and in vivo a hyper-depolymerizing {beta}-actin mutant displays a heightened association with RPA and the same dysfunctional replication phenotypes as loss of ANs/NPFs, which contrasts with the phenotype of a hyper-polymerizing {beta}-actin mutant. Thus, we identify components of actin polymerization pathways that are essential for preventing ectopic nucleolytic degradation of perturbed forks by modulating RPA activity.

molecular biology↗

bioRxiv2023

A bioinformatics screen reveals Hox and chromatin remodeling factors at the Drosophila histone locus

Cells orchestrate histone biogenesis with strict temporal and quantitative control. To efficiently regulate histone biogenesis, the repetitive Drosophila melanogaster replication-dependent histone genes are arrayed and clustered at a single locus. Regulatory factors concentrate in a nuclear body known as the histone locus body (HLB), which forms around the locus. Historically, HLB factors are largely discovered by chance, and few are known to interact directly with DNA. It is therefore unclear how the histone genes are specifically targeted for unique and coordinated regulation. To expand the list of known HLB factors, we performed a candidate-based screen by mapping 30 publicly available ChIP datasets and 27 factors to the Drosophila histone gene array. We identified novel transcription factor candidates, including the Drosophila Hox proteins Ultrabithorax, Abdominal-A and Abdominal-B, suggesting a new pathway for these factors in influencing body plan morphogenesis. Additionally, we identified six other transcription factors that target the histone gene array: JIL-1, Hr78, the long isoform of fs(1)h as well as the generalized transcription factors TAF-1, TFIIB, and TFIIF. Our foundational screen provides several candidates for future studies into factors that may influence histone biogenesis. Further, our study emphasizes the powerful reservoir of publicly available datasets, which can be mined as a primary screening technique.

molecular biology↗

bioRxiv2022

High-resolution app data reveal sustained increases in recreational fishing effort in Europe during and after COVID-19 lockdowns

It is manifest that COVID-19 lockdowns extensively impacted human interactions with natural ecosystems. One example is recreational fishing, an activity which involves nearly 1 in 10 people in developed countries. Fishing licence sales and direct observations at popular angling locations suggest that recreational fishing effort increased substantially during lockdowns. However, the extent and duration of this increase remain largely unknown due to a lack of objective data. We used four years (2018 to 2021) of anonymous, high-resolution data from a personal fish-finder device to explore the impact of COVID-19 lockdowns on recreational fishing effort in four European countries (Lithuania, the Czech Republic, Denmark, and Germany). We show that device use and, by extension, angling effort increased 1.2-3.8 fold during March-May 2020 and remained elevated even at the end of 2021 in all countries except Denmark. Fishing during the first lockdown also became more frequent during weekdays. Statistical models with the full set of fixed (weekdays, lockdown, population) and random (season, year, administrative unit) factors typically explained 50-70% of the variation, suggesting that device use and angling effort were relatively consistent and predictable through space and time. Our study demonstrates that recreational fishing behaviour can change substantially and rapidly in response to societal shifts, with profound ecological, human well-being and economic implications. We also show the potential of angler devices and smartphone applications to supply data for high-resolution fishing effort analysis and encourage more extensive science and industry collaborations to take advantage of this information. Significance statementRecreational fishing is a popular and widespread activity with ecological, social and economic impacts, though problematic to assess and manage due to a paucity of information regarding effort and catch. Here, we use high-resolution data from a personal angler sonar device to show how the COVID-19 pandemic changed angler behaviour and fishing effort across Europe. We demonstrate that angling effort doubled and remained higher at the end of 2021 than before the first lockdowns. Such rapid and profound changes could have significant consequences for aquatic ecosystems, possibly requiring new management approaches. We encourage the adoption of novel data from angler devices, citizen science, and more active science-industry collaborations to improve recreational fishing assessment and management.

ecology↗

bioRxiv2022

RBPMS promotes contractile smooth muscle splicing and alters phenotypic behaviour of human embryonic stem cell derived vascular smooth muscle cells.

Differentiated Vascular Smooth Muscle Cells (VSMCs) express a unique network of splice isoforms (smooth muscle specific alternative splicing - SM-AS) in functionally critical genes including those comprising the contractile machinery. We previously described RNA Binding Protein Multiple Splicing (RBPMS) as a potent driver of contractile, aortic tissue like SM-AS in VSMCs using rodent models. What is unknown is how RBPMS affects VSMC phenotype and behaviour. Here, we use human embryonic stem cell-derived VSMCs (hES-VSMCs) to dissect the role of RBPMS in SM-AS in human cells and determine the impact on VSMC phenotypic properties. hES-VSMCs are inherently immature and display only partially differentiated SM-AS patterns while RBPMS levels are undetectable endogenously. Hence, we used an over-expression system and found that RBPMS induces SM-AS patterns in hES-VSMCs akin to the contractile tissue VSMC splicing patterns in multiple events. We present in silico and experimental findings that support RBPMS splicing activity as mediated through direct binding and via functional cooperativity with splicing factor RBFOX2 on a significant subset of targets. Finally, we demonstrate that RBPMS is capable of altering the motility and the proliferative properties of hES-VSMCs to mimic a more differentiated state. Overall, this study emphasizes a critical splicing regulatory role for RBPMS in human VSMCs and provides evidence of phenotypic modulation by RBPMS.

molecular biology↗

bioRxiv2022

Does the Ice Age legacy end in Central Europe? The shrinking distributions of glacial relict crustaceans

O_LIGlacial relict mysid and amphipod crustaceans are characterised by their affinity for cold and well-oxygenated waters and inability to disperse upstream or by external agents. These crustaceans occur in large and deep lakes of Northern and Central Europe and North America, their distributions shaped by glaciation events. In Europe, along the southern edge of their distribution (Germany, Poland, Lithuania, Belarus), glacial relict crustaceans are threatened by eutrophication and global warming. C_LIO_LIThis study assesses the status of three glacial relict malacostracan species in Lithuania; the amphipods Monoporeia affinis and Pallaseopsis quadrispinosa, and mysid Mysis relicta, and models their abundance as a function of environmental variables and the presence of invasive Ponto-Caspian mysids and amphipods. C_LIO_LIOur results revealed that M. affinis is likely extinct in the country, whereas M. relicta was found in only 9 out of 16 locations from which it was previously recorded. The distribution of P. quadrispinosa appears to be shrinking. C_LIO_LILake depth and water flowthrough intensity were significantly and positively associated with the relative abundance of relict mysids and amphipods, but no association was found with lake size or the presence of invasive Ponto-Caspian crustaceans. C_LIO_LIWe conclude that urgent action to mitigate the effects of nutrient run-off is needed to improve the status of glacial relict and other species that require good water quality. We also propose the re-introduction of glacial relict species in Lake Dr[u]k[s]iai, where they went extinct during the operation of the Ignalina nuclear power plant that heated the lake, but where deep-water environmental conditions have improved following the powerplant closure in 2010. C_LI

ecology↗

bioRxiv2022

The Effect of Polymer Length in Phase Separation

Understanding the thermodynamics that drives liquid-liquid phase separation (LLPS) is quite important given the many numbers of diverse biomolecular systems undergoing this phenomenon. Regardless of the diversity, the processes underlying the formation of condensates exhibit physical similarities. Many studies have focused on condensates of long polymers, but very few systems of short polymer condensates have been observed and yet studied. Here we study a short polymer system of various lengths of poly-Adenine RNA and peptide formed by the RGRGG sequence repeats to understand the underlying thermodynamics of LLPS. We carried out MD simulations using the recently developed COCOMO coarse-grained (CG) model which revealed the possibility of condensates for lengths as short as 5-10 residues, which was then confirmed by experiment, making this one of the smallest LLPS systems yet observed. Condensation depends on polymer length and concentration, and phase boundaries were identified. A free energy model was also developed. Results show that the length dependent condensation is driven solely by entropy of confinement and identifies a negative free energy (-{Delta}G) of phase separation, indicating the stability of the condensates. The simplicity of this system will provide the basis for understanding more biologically realistic systems.

biophysics↗

bioRxiv2022

Comprehensive single-cell transcriptomic analysis of embryonic melanoblasts uncovers lineage-specific mechanisms of melanoma metastasis and therapy resistance

Melanoma plasticity, driven by phenotype state switching, underlies clinically relevant traits such as metastasis and therapy resistance. As melanoma progression is thought to recapitulate aspects of neural crest cell (NCC) development, understanding embryonic melanocyte specification and lineage fate decisions of closely related NCCs may illuminate the pathways co-opted during disease evolution. Here, we use a mouse model to isolate and sequence Dopachrome tautomerase (Dct) expressing NCCs, the precursors of melanocytes, at two key developmental stages. We classify these lineages and devise a Developmental Gene Module (DGM) scoring system to interrogate lineage state switching in melanoma samples. In bulk transcriptomes, activation of DGMs representing embryonic Schwann Cell Precursors (SCPs)--multipotent stem cells--in patient tumors predicts poor response to immune checkpoint inhibitors (ICI). Co-activation of SCP and Mesenchymal-like (Mes.) modules further correlates with resistance to MAPK inhibitors. Notably, single-cell analyses reveal that melanoma cells can simultaneously express multiple DGMs, forming "hybrid" states. Cells in a hybrid Neural/SCP state are enriched in early metastasis and ICI-resistant tumors and are insensitive to inflammatory stimuli. We demonstrate that targeting Hdac2, a histone deacetylase associated with this Neural/SCP hybrid state, promotes a mesenchymal-like state switch, remodels the tumor microenvironment, and sensitizes melanoma cells to TNF and tumors to ICI therapy. Our methodology thus reveals dynamic patterns of lineage state switching correlated with melanoma tumor evolution to drive insight into new therapeutic targets. TeaserNewly identified melanoblast cell states are reawakened in metastasizing and therapy-resistant melanomas.

cancer biology↗

bioRxiv2022

Using pressure-driven flow systems to evaluate laser speckle contrast imaging

SignificanceMicrofluidic flow phantom studies are commonly used for characterizing the performance of laser speckle contrast imaging (LSCI) instruments. The selection of the flow control system is critical for the reliable generation of flow during testing. The majority of recent LSCI studies using microfluidics used syringe pumps for flow control. AimWe quantified the uncertainty in flow generation for a syringe pump and a pressure-regulated flow system. We then assessed the performance of both LSCI and multi-exposure speckle imaging (MESI) using the pressure-regulated flow system across a range of flow speeds. ApproachThe syringe pump and pressure-regulated flow systems were evaluated during stepped flow profile experiments in a microfluidic device using an inline flow sensor. The uncertainty associated with each flow system was calculated and used to determine the reliability for instrument testing. The pressure-regulated flow system was then used to characterize the relative performance of LSCI and MESI during stepped flow profile experiments while using the inline flow sensor as reference. ResultsThe pressure-regulated flow system produced much more stable and reproducible flow outputs compared to the syringe pump. The expanded uncertainty for the syringe pump was 8-20x higher than that of the pressure-regulated flow system across the tested flow speeds. Using the pressure-regulated flow system, MESI outperformed single-exposure LSCI at all flow speeds and closely mirrored the flow sensor measurements, with average errors of 4.6 {+/-} 2.6% and 15.7 {+/-} 4.6%, respectively. ConclusionsPressure-regulated flow systems should be used instead of syringe pumps when assessing the performance of flow measurement techniques with microfluidic studies. MESI offers more accurate relative flow measurements than traditional LSCI across a wide range of flow speeds.

bioengineering↗