Biology subjects

Gibson, I.

Publications and source records attributed to Gibson, I..

3 recordsLinked to original sources

bioRxiv2025

Using Beads as a Focus Fiduciary to Aid Software-based Autofocus Accuracy in Microscopy

Brightfield microscopy is an ideal application for studying live cell systems in a minimally invasive manner. This is advantageous in long-term experiments to study dynamic cellular processes such as stress response. Depending on the sample type and preparation, the inherent qualities of brightfield microscopy, being very low contrast, can contribute to technical issues such as focal drift, sequencing lags, and complete failure of software autofocus systems. Here, we describe the use of microbeads as a focus aid for long-term live cell imaging to address these autofocus issues. This protocol is inexpensive to implement, without extensive additional sample preparation and can be used to capture focused images of transparent cells in a label-free manner. To validate this protocol, a widefield inverted microscope was used with software-based autofocus to image overnight in time-lapse format, demonstrating the use of the beads to prevent focal drift in long-term experiments. This improves autofocus accuracy on relatively inexpensive microscopes without using hardware-based focus aids. To validate this protocol, the KNIME logistics software was used to train a random forest model to perform binary image classification. Key featuresO_LIlabel-free live cell imaging in time-lapse format C_LIO_LITroubleshooting software autofocus for brightfield mode C_LI Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/643916v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1038c25org.highwire.dtl.DTLVardef@1e5f175org.highwire.dtl.DTLVardef@14f394corg.highwire.dtl.DTLVardef@3abca3_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Multiscale genetic architecture of donor-recipient differences reveals intronic LIMS1 locus mismatches associated with long-term renal transplant survival

BackgroundLong-term kidney allograft survival remains suboptimal. Emerging evidence indicates donor-recipient (D-R) mismatches outside of human leukocyte antigens (HLA) contribute to graft survival but mechanisms remain unclear, specifically for those mismatches within intronic regions. MethodsWe analyzed genome-wide SNP data of D-R pairs from two well-phenotyped kidney transplant cohorts (median follow-up ~1800 days), Genomics of Chronic Allograft Rejection (GoCAR; n=385) and Clinical Trials in Organ Transplantation 1/17 (CTOT1/17; n=146), quantifying genetic mismatches outside of HLA for every D-R pair at variant, gene, and genome-wide scales. ResultsUnbiased genome-wide screen of GoCAR D-R pairs uncovered the LIMS1 locus as the topranked candidate where D-R mismatches associated with death censored graft loss (DCGL). Independent of HLA, a previously unreported relationship between mismatches at a highly linked, intronic haplotype of 30 SNPs was seen as associated with DCGL, with confirmatory association in intra-ancestry D-Rs. Validation testing within the CTOT-01/17 showed similar associations with DCGL. Haplotype D-R mismatches showed a dosage effect, and the introduction of minor alleles in the donor to major allele-carrying recipients showed a greater risk of DCGL. Both the new LIMS1 haplotype and the previously reported LIMS1 SNP rs893403 are expression quantitative trait loci (eQTL) for the gene GCC2 in recipient immune cells, without alterations in GCC2 or LIMS1 coding sequences. Transcriptome enrichment analyses performed on whole blood and within multiple T cell subsets demonstrated significant associations of GCC2 gene, and of either allelic locus, with regulation of TGF-beta-SMAD signaling, implying a role in Treg function and association with rejection. ConclusionsOur analysis unravels intronic non-HLA SNP mismatches within LIMS1 that do not induce protein sequence variation but associate with DCGL. By acting as cis-eQTLs for GCC2 expression, these SNPs modulates TGF-beta signaling and T cell function, associating with immune events and graft outcomes. The findings have clinical implications for risk assessment and individualized therapy in kidney transplant recipients.

genetics↗

bioRxiv2021

Forward genetics combined with unsupervised classifications identified zebrafish mutants affecting biliary system formation.

Impaired formation of the biliary network can lead to congenital cholestatic liver diseases; however, the genes responsible for proper biliary system formation and maintenance have not been fully identified. Combining computational network structure analysis algorithms with a zebrafish forward genetic screen, we identified 24 new zebrafish mutants that display impaired intrahepatic biliary network formation. Complementation tests suggested that these 24 mutants affect 24 different genes. We applied unsupervised clustering algorithms to classify the recovered mutants into three classes unbiasedly. Further computational analyses revealed that each of the recovered mutations in these three classes shows a unique effect on node subtype composition and connection property distribution of the intrahepatic biliary network. Besides, we found that most recovered mutations are viable. In those mutant fish, biliary network phenotypes persist into adulthood, which themselves are good animal models to study chronic cholestatic liver diseases. Altogether, this study provides unique genetic and computational toolsets that advance our understanding of the molecular pathways leading to biliary system malformation and cholestatic liver diseases.

genetics↗