Biology subjects

Rowe, D.

Publications and source records attributed to Rowe, D..

8 recordsLinked to original sources

bioRxiv2026

High-Throughput, Multiplex Immunofluorescence for the Computer-automated Immunophenotyping of Mycosis Fungoides

The diagnosis of Mycosis Fungoides (MF) is difficult and often delayed, exacerbated by the constraint of conventional immunohistochemistry (IHC) to analyze only one antigen per tissue section, often necessitating repeat biopsies and extensive workups. We sought to validate a high-throughput Multiplex Immunofluorescence (MIF) method, coupled with computer-automated image analysis, to generate comprehensive immunophenotyping data from a single formalin-fixed, paraffin-embedded (FFPE) biopsy. We applied an 11-biomarker MIF panel across 18 archived skin specimens (9 MF/TCR clonality positive and 9 control/TCR clonality negative). Initial validation confirmed that MIF antigen expression and spatial localization were concordant with sequential IHC-stained sections. Whole slide image stacks were analyzed using both computer-assisted and fully computer-automated pipelines. Both methods successfully delineated immunophenotypic differences. MF specimens showed a significant expansion of hematopoietic cells and proliferative T-lymphocytes compared to controls. Crucially, MF tissues also exhibited a significant increase in the percentage of atypical T-lymphocytes. Our results validate the potential of MIF to obtain comprehensive, high-dimensional diagnostic information from a single tissue section. Integration with computer-automated analysis offers a scalable, high-throughput platform that can significantly aid in the timely and accurate diagnosis of cutaneous lymphomas.

pathology↗

bioRxiv2024

Hair follicle-resident progenitor cells are a major cellular contributor to heterotopic subcutaneous ossifications in a mouse model of Albright hereditary osteodystrophy

Heterotopic ossifications (HOs) are the pathologic process by which bone inappropriately forms outside of the skeletal system. Despite HOs being a persistent clinical problem in the general population, there are no definitive strategies for their prevention and treatment due to a limited understanding of the cellular and molecular mechanisms contributing to lesion development. One disease in which the development of heterotopic subcutaneous ossifications (SCOs) leads to morbidity is Albright hereditary osteodystrophy (AHO). AHO is caused by heterozygous inactivation of GNAS, the gene that encodes the -stimulatory subunit (Gs) of G proteins. Previously, we had shown using our laboratorys AHO mouse model that SCOs develop around hair follicles (HFs). Here we show that SCO formation occurs due to inappropriate expansion and differentiation of HF-resident stem cells into osteoblasts. We also show in AHO patients and mice that Secreted Frizzled Related Protein 2 (SFRP2) expression is upregulated in regions of SCO formation and that elimination of Sfrp2 in male AHO mice exacerbates SCO development. These studies provide key insights into the cellular and molecular mechanisms contributing to SCO development and have implications for potential therapeutic modalities not only for AHO patients but also for patients suffering from HOs with other etiologies.

cell biology↗

bioRxiv2024

Combining population genomics and transcriptomics to identify signatures of metal tolerance in brown trout inhabiting metal-polluted rivers

Industrial pollution is a major driver of ecosystem degradation, but it can also act as a driver of contemporary evolution. As a result of intense mining activity during the Industrial Revolution, several rivers across the southwest of England are polluted with high concentrations of metals. Despite the documented negative impacts of ongoing metal pollution, brown trout (Salmo trutta L.) survive and thrive in many of these metal-impacted rivers. We used population genomics, transcriptomics, and metal burdens to investigate the genomic and transcriptomic signatures of potential metal tolerance. RADseq analysis of six populations (originating from three metal-impacted and three control rivers) revealed strong genetic substructuring between impacted and control populations. We identified selection signatures at 122 loci, including genes related to metal homeostasis and oxidative stress. Trout sampled from metal-impacted rivers exhibited significantly higher tissue concentrations of cadmium, copper, nickel, and zinc, which remained elevated after 11 days in metal-free water. After depuration, we used RNAseq to quantify gene expression differences between metal-impacted and control trout, identifying 2,042 differentially expressed genes (DEGs) in the gill, and 311 DEGs in the liver. Transcriptomic signatures in the gill were enriched for genes involved in ion transport processes, metal homeostasis, oxidative stress, hypoxia and response to xenobiotics. Our findings reveal shared genomic and transcriptomic pathways involved in detoxification, oxidative stress responses, and ion regulation. Overall, our results demonstrate the diverse effects of metal pollution in shaping both neutral and adaptive genetic variation, whilst also highlighting the potential role of constitutive gene expression in promoting metal tolerance.

evolutionary biology↗

bioRxiv2024

vSPACE: Exploring Virtual Spatial Representation of Articular Chondrocytes at the Single-Cell Level

Single cell RNA sequencing technology has been dramatically changing how gene expression studies are performed. However, its use has been limited to identifying subtypes of cells by comparing cells gene expression levels in an unbiased manner to produce a 2D plot (e.g., UMAP/tSNE). We developed a new method of placing cells in 2D space. This system, called vSPACE, shows a virtual spatial representation of scRNAseq data obtained from human articular cartilage by emulating the concept of spatial transcriptomics technology, but virtually. This virtual 2D plot presentation of human articular cartage cells generates several zonal distribution patterns, in one or multiple genes at a time, reveling patterns that scientists can appreciate as imputed spatial distribution patterns along the zonal axis. The discovered patterns are explainable and remarkably consistent across all six healthy doners despite their respectively different clinical variables (age and sex), suggesting the confidence of the discovered patterns.

bioinformatics↗

bioRxiv2023

Natural genomic variation in rice blast genomes is associated with specific heterochromatinmodifications

Genome organization in eukaryotes exhibits non-random patterns tied to transcription, replication, and chromatin. However, the driving forces across these processes, and their impacts on genome evolution remain unclear. To address this, we analyzed sequence data from 86 Magnaporthe oryzae isolates, a globally important plant pathogenic fungus, alongside new reference genomes, to investigate DNA sequence variation and the epigenome. Histone modification profiles were obtained through genome-wide chromatin immunoprecipitation-sequencing of the four new reference strains, which revealed that repressive histone marks (H3K27me3, H3K9me3) were associated with SNP and INDEL frequency. Densely grouped SNPs were found to reside in heterochromatin, often outside transposable elements, highlighting the link between heterochromatin and DNA variation. Even when controlling for selection, silent SNP frequency/kb was higher in H3K27me3-associated genes. Effector genes, key to pathogenicity, also displayed this trend. Comparing the reference strains, euchromatic regions were often syntenic, while heterochromatic regions trended towards non-syntenic. Heterochromatin emerged as a major factor associated with diverse DNA variations in M. oryzae populations, even when accounting for selective pressure. This underscores heterochromatins pivotal role in shaping genetic diversity in these mainly asexually reproducing fungi.

evolutionary biology↗

bioRxiv2021

CRISPR-Cas12a induced DNA double-strand breaks are repaired by locus-dependent and error-prone pathways in a fungal pathogen

CRISPR-Cas mediated genome engineering has revolutionized functional genomics. However, basic questions remain regarding the mechanisms of DNA repair following Cas-mediated DNA cleavage. We developed CRISPR-Cas12a ribonucleoprotein genome editing in the fungal plant pathogen, Magnaporthe oryzae, and found frequent donor DNA integration despite the absence of long sequence homology. Interestingly, genotyping from hundreds of transformants showed that frequent non-canonical DNA repair outcomes predominated the recovered genome edited strains. Detailed analysis using sanger and nanopore long-read sequencing revealed five classes of DNA repair mutations, including single donor DNA insertions, concatemer donor DNA insertions, large DNA deletions, deletions plus donor DNA insertions, and infrequently we observed INDELs. Our results show that different error-prone DNA repair pathways resolved the Cas12a-mediated double-strand breaks (DSBs) based on the DNA sequence of edited strains. Furthermore, we found that the frequency of the different DNA repair outcomes varied across the genome, with some tested loci resulting in more frequent large-scale mutations. These results suggest that DNA repair pathways provide preferential repair across the genome that could create biased genome variation, which has significant implications for genome engineering and the genome evolution in natural populations.

microbiology↗

bioRxiv2021

Parental origin of Gsα inactivation differentially affects bone remodeling in a mouse model of Albright hereditary osteodystrophy

Albright hereditary osteodystrophy (AHO) is caused by heterozygous inactivation of GNAS, a complex locus that encodes the alpha-stimulatory subunit of GPCRs (Gs) in addition to NESP55 and XLs due to alternative first exons. AHO skeletal manifestations include brachydactyly, brachymetacarpia, compromised adult stature, and subcutaneous ossifications. AHO patients with maternally-inherited GNAS mutations develop pseudohypoparathyroidism type 1A (PHP1A) with resistance to multiple hormones that mediate their actions through GPCRs requiring Gs (eg., PTH, TSH, GHRH, calcitonin) and severe obesity. Paternally-inherited GNAS mutations cause pseudopseudohypoparathyroidism (PPHP), in which patients have AHO skeletal features but do not develop hormonal resistance or marked obesity. These differences between PHP1A and PPHP are caused by tissue-specific reduction of paternal Gs expression. Previous reports in mice have shown loss of Gs causes osteopenia due to impaired osteoblast number and function and suggest AHO patients could display evidence of reduced bone mineral density (BMD). However, we previously demonstrated PHP1A patients display normal-increased BMD measurements without any correlation to body mass index or serum PTH. Due to these observed differences between PHP1A and PPHP, we utilized our laboratorys AHO mouse model to address whether Gs heterozygous inactivation by the targeted disruption of exon 1 of Gnas differentially affects bone remodeling based on the parental inheritance of the mutation. Mice with paternally-inherited (GnasE1+/-p) and maternally-inherited (GnasE1+/-m) mutations displayed reductions in osteoblasts along the bone surface compared to wildtype. GnasE1+/-p mice displayed reduced cortical and trabecular bone parameters due to impaired bone formation and excessive bone resorption. GnasE1+/-m mice however displayed enhanced bone parameters due to increased osteoblast activity and normal bone resorption. These distinctions in bone remodeling between GnasE1+/-p and GnasE1+/-m mice appear to be secondary to changes in the bone microenvironment driven by calcitonin-resistance within GnasE1+/-m osteoclasts and therefore warrant further studies into understanding how Gs influences osteoblast-osteoclast coupling interactions.

cell biology↗

bioRxiv2018

Impacts of Simultaneous Multislice Acquisition on Sensitivity and Specificity in fMRI

Simultaneous multislice (SMS) acquisition can be used to decrease the time between acquisition of fMRI volumes, which can increase sensitivity by facilitating the removal of higher-frequency artifacts and boosting effective sample size. The technique requires an additional processing step in which the slices are separated, or unaliased, to recover the whole brain volume. However, this may result in signal \"leakage\" between aliased locations, i.e., slice \"leakage,\" and lead to spurious activation (decreased specificity). SMS can also lead to noise amplification, which can reduce the benefits of decreased repetition time. In this study, we evaluate the original slice-GRAPPA (no leak block) reconstruction algorithm and acquisition scenarios used in the young adult Human Connectome Project (HCP), as well as split slice-GRAPPA (leak block). In addition to slice leakage, signal leakage can result from spatial smoothing, i.e., smoothing leakage, which leads to inflated regions of activation. Previous studies have generally found that SMS acquisition results in higher test statistics and/or a greater number of activated voxels. Here, we use simulations to disentangle this phenomenon into true positives (sensitivity) and false positives (decreased specificity). Slice leakage was greatly decreased by split slice-GRAPPA. Noise amplification was decreased by using moderate acceleration factors (AF = 4). We examined slice leakage in unprocessed fMRI motor task data from the HCP, which used the original slice-GRAPPA. When data were smoothed, we found evidence of slice leakage in some, but not all, subjects. We also found evidence of SMS noise amplification in unprocessed task and processed resting-state HCP data.

neuroscience↗