Biology subjects

Witt, E.

Publications and source records attributed to Witt, E..

7 recordsLinked to original sources

bioRxiv2025

Enhancing HIPEC for Ovarian Cancer using Adjunctive Biomaterials

Ovarian cancer is one of the most lethal gynecological malignancies, with high mortality rates primarily due to late-stage diagnoses and extensive peritoneal metastases. Despite improvements in surgical and chemotherapeutic treatments, the prognosis for advanced ovarian cancer remains poor, highlighting the urgent need for innovative therapeutic approaches. Hyperthermic intraperitoneal chemotherapy (HIPEC) has emerged as a promising treatment, delivering heated chemotherapeutic agents directly into the peritoneal cavity post-cytoreductive surgery. However, HIPEC adoption is limited by three critical complications: suboptimal therapeutic efficacy in resistant tumors; abdominal adhesion formation; and systemic toxicity, including cisplatin-induced nephrotoxicity. This study investigates carbon monoxide gas- entrapping materials (CO-GEMs) as a novel multifunctional adjunctive therapy to address the three HIPEC limitations simultaneously. CO-GEMs effectively encapsulate and deliver carbon monoxide, leveraging the differential effects of CO in cancerous where it has been shown to reduce tumor burden. In ovarian cancer models, CO-GEMs significantly enhanced cisplatin efficacy, reducing the metastatic tumor burden by 46.6% through the downregulation of drug resistance pathways, including the IL-17, TNF, and NF-{kappa}B pathways; ECM receptor interaction, and VEGF signaling. CO-GEMs also prevented peritoneal adhesion formation by suppressing inflammatory cell infiltration and collagen deposition, with a significant reduction in adhesion severity scores. Additionally, enteral CO-GEMs provided significant nephroprotection against cisplatin-induced acute kidney injury, as demonstrated by reduced blood urea nitrogen levels. CO-GEMs represent a promising innovation that simultaneously improves HIPEC therapeutic efficacy, prevents surgical complications, and reduces systemic toxicity. This multifunctional approach addresses multiple clinical limitations of HIPEC, potentially transforming treatment outcomes for patients with advanced ovarian cancer through an enhanced therapeutic index and improved safety profile.

bioengineering↗

bioRxiv2023

Multiplexed live-cell imaging for drug responses in patient-derived organoid models of cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to simultaneously quantitate cell health and apoptosis. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer. SUMMARYPatient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

cancer biology↗

bioRxiv2023

Early resource scarcity causes cortical astrocyte enlargement and sex-specific changes in the orbitofrontal cortex transcriptome in adult rats

Astrocyte morphology affects function, including the regulation of glutamatergic signaling. This morphology changes dynamically in response to the environment. However, how early life manipulations alter adult cortical astrocyte morphology is underexplored. Our lab uses brief postnatal resource scarcity, the limited bedding and nesting (LBN) manipulation, in rats. We previously found that LBN promotes later resilience to adult addiction-related behaviors, reducing impulsivity, risky decision-making, and morphine self-administration. These behaviors rely on glutamatergic transmission in the medial orbitofrontal (mOFC) and medial prefrontal (mPFC) cortex. Here we tested whether LBN changed astrocyte morphology in the mOFC and mPFC of adult rats using a novel viral approach that, unlike traditional markers, fully labels astrocytes. Prior exposure to LBN causes an increase in the surface area and volume of astrocytes in the mOFC and mPFC of adult males and females relative to control-raised rats. We next used bulk RNA sequencing of OFC tissue to assess transcriptional changes that could increase astrocyte size in LBN rats. LBN caused mainly sex-specific changes in differentially expressed genes. However, Park7, which encodes for the protein DJ-1 that alters astrocyte morphology, was increased by LBN across sex. Pathway analysis revealed that OFC glutamatergic signaling is altered by LBN in males and females, but the gene changes in that pathway differed across sex. This may represent a convergent sex difference where glutamatergic signaling, which affects astrocyte morphology, is altered by LBN via sex-specific mechanisms. Collectively, these studies highlight that astrocytes may be an important cell type that mediates the effect of early resource scarcity on adult brain function.

neuroscience↗

bioRxiv2021

Transcriptional and mutational signatures of the aging germline

Aging is a complex biological process that is accompanied by changes in gene expression and mutational load. In many species, including humans, older fathers pass on more paternally-derived de novo mutations; however, the cellular basis and cell types driving this pattern are still unclear. To explore the root causes of this phenomenon, we performed single-cell RNA-sequencing (scRNA-seq) on testes from young and old male Drosophila, as well as genomic sequencing (DNA-seq) on somatic tissues from the same flies. We found that early germ cells from old and young flies enter spermatogenesis with similar mutational loads, but older flies are less able to remove mutations during spermatogenesis. Mutations in old cells may also increase during spermatogenesis. Our data reveal that old and young flies have distinct mutational biases. Many classes of genes show increased post-meiotic expression in the germlines of older flies. Late spermatogenesis-enriched genes have higher dN/dS than early spermatogenesis-enriched genes, supporting the hypothesis that late spermatogenesis is a source of evolutionary innovation. Surprisingly, young fly enriched genes show higher dN/dS than old fly enriched genes. Our results provide novel insights into the role of the germline in de novo mutation.

evolutionary biology↗

bioRxiv2021

Single-cell RNA-sequencing reveals pre-meiotic X-chromosome dosage compensation in Drosophila testis

Dosage compensation (DC) is a mechanism by which X chromosome transcription is equalized in the somatic cells of both males and females. In male fruit flies, expression levels of the X-chromosome are increased two-fold to compensate for their single X chromosome. In testis, dosage compensation is thought to cease during meiosis, however, the timing and degree of the resulting transcriptional suppression is difficult to separate from global meiotic downregulation of each chromosome. To address this, we analyzed testis single-cell RNA-sequencing (scRNA-seq) data from two Drosophila melanogaster strains. We found evidence that the X chromosome is equally transcriptionally active as autosomes in somatic and pre-meiotic cells, and less transcriptionally active than autosomes in meiotic and post-meiotic cells. In cells experiencing dosage compensation, close proximity to MSL (male-specific lethal) chromatin entry sites (CES) correlates with increased X chromosome transcription. We found low or undetectable level of germline expression of most msl genes, mle, roX1 and roX2 via sequencing or RNA-FISH, and no evidence of germline nuclear roX1/2 localization. Our results suggest that, although DC occurs in somatic and premeiotic germ cells in Drosophila testis, there might be non-canonical factors involved in the dosage compensation. The single-cell expression patterns and enrichment statistics of detected genes can be explored interactively in our database: https://zhao.labapps.rockefeller.edu/gene-expr/.

genetics↗

bioRxiv2020

Transcription factors drive opposite relationships between gene age and tissue specificity in male and female Drosophila gonads

Evolutionarily young genes are usually preferentially expressed in the testis across species. While it is known that older genes are generally more broadly expressed than younger genes, the properties that shaped this pattern are unknown. Older genes may gain expression across other tissues uniformly, or faster in certain tissues than others. Using Drosophila gene expression data, we confirmed previous findings that younger genes are disproportionately testis-biased and older genes are disproportionately ovary-biased. We found that the relationship between gene age and expression is stronger in the ovary than any other tissue, and weakest in testis. We performed ATAC-seq on Drosophila testis and found that while genes of all ages are more likely to have open promoter chromatin in testis than in ovary, promoter chromatin alone does not explain the ovary-bias of older genes. Instead, we found that upstream transcription factor (TF) expression is highly predictive of gene expression in ovary, but not in testis. In ovary, TF expression is more predictive of gene expression than open promoter chromatin, whereas testis gene expression is similarly influenced by both TF expression and open promoter chromatin. We propose that the testis is uniquely able to expresses younger genes controlled by relatively few TFs, while older genes with more TF partners are broadly expressed with peak expression most likely in ovary. The testis allows widespread baseline expression that is relatively unresponsive to regulatory changes, whereas the ovary transcriptome is more responsive to trans-regulation and has a higher ceiling for gene expression.

evolutionary biology↗

bioRxiv2019

Testis single-cell RNA-seq reveals the dynamics of de novo gene transcription and germline mutational bias in Drosophila

The testis is a peculiar tissue in many respects. It shows patterns of rapid gene evolution and provides a hotspot for the origination of genetic novelties such as de novo genes, duplications and mutations. To investigate the expression patterns of genetic novelties across cell types, we performed single-cell RNA-sequencing of adult Drosophila testis. We found that new genes were expressed in various cell types, the patterns of which may be influenced by their mode of origination. In particular, lineage-specific de novo genes are commonly expressed in early spermatocytes, while young duplicated genes are often bimodally expressed. Analysis of germline substitutions suggests that spermatogenesis is a highly reparative process, with the mutational load of germ cells decreasing as spermatogenesis progresses. By elucidating the distribution of genetic novelties across spermatogenesis, this study provides a deeper understanding of how the testis maintains its core reproductive function while being a hotbed of evolutionary innovation.

evolutionary biology↗