Biology subjects

Crane, M. M.

Publications and source records attributed to Crane, M. M..

7 recordsLinked to original sources

bioRxiv2020

Methods for High-Fidelity Spectral Data Collection for Generating Ground Truth Data for Simulated Tissues

Our research is focused on creating and simulating hyper-realistic artificial human tissue analogues. Generation and simulation of macroscopic biological material depends upon accurate ground-truth data on spectral properties of materials. Here, we developed methods for high fidelity spectral data collection using two differently colored simulated skin tissue samples and a portable spectral imaging camera. Using the standard procedure, we developed, we quantified the reproducibility of the spectral image signatures of the two synthetic skin samples under natural and artificial lighting conditions commonly found in clinical settings. We found high coefficients of determination for all measures taken under the same lighting. As expected, we found the spectral image signature of each sample was dependent on the illumination source. Our results confirm that illumination spectra data should be included with spectral image data. The high-fidelity methods for spectral image data collection we developed here should facilitate accurate collection of spectral image signature data for gross biological samples and synthetic materials collected under the same illumination source.

bioengineering↗

bioRxiv2020

Loss of vacuolar acidity results in iron sulfur cluster defects and divergent homeostatic responses during aging in Saccharomyces cerevisiae

The loss of vacuolar/lysosomal acidity is an early event during aging that has been linked to mitochondrial dysfunction. However, it is unclear how loss of vacuolar acidity results in age-related dysfunction. Through unbiased genetic screens, we determined that increased iron uptake can suppress the mitochondrial respiratory deficiency phenotype of yeast vma mutants, which have lost vacuolar acidity due to genetic disruption of the vacuolar ATPase proton pump. Yeast vma mutants exhibited nuclear localization of Aft1, which turns on the iron regulon in response to iron sulfur cluster (ISC) deficiency. This led us to find that loss of vacuolar acidity with age in wildtype yeast causes ISC defects and a DNA damage response. Using microfluidics to investigate aging at the single cell level, we observe grossly divergent trajectories of iron homeostasis within an isogenic and environmentally homogeneous population. One subpopulation of cells fails to mount the expected compensatory iron regulon gene expression program, and suffers progressively severe ISC deficiency with little to no activation of the iron regulon. In contrast, other cells show robust iron regulon activity with limited ISC deficiency, which allows extended passage and survival through a period of genomic instability during aging. These divergent trajectories suggest that iron regulation and ISC homeostasis represent a possible target for aging interventions.

molecular biology↗

bioRxiv2019

A physicochemical roadmap of yeast replicative aging

Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding, are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show the macromolecular crowding changes less in longer-lived cells in contrast to shorter-lived cells. While the average pH and crowding levels change only modestly with aging, we observe drastic changes in organellar volume, leading to crowding on the {micro}m scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively-aged yeast cells.

biophysics↗

bioRxiv2019

Microbiome Stability with Chronic SIV Infection in AIDS-resistant Sooty Mangabeys

Sooty mangabeys (SMs) are a natural host species of simian immunodeficiency virus (SIV) and avoid acquired immune deficiency syndrome (AIDS) despite persistently high viral loads, making them a pivotal research model for HIV pathogenesis. Unlike pathogenic SIV infection of macaque species, or HIV infection of humans, SIV-infected SMs maintain gastrointestinal barrier integrity. Here, we characterize the gastrointestinal bacterial microbiota of SIV-infected and uninfected SMs and perform a comparative analysis of diet-matched, rhesus macaques (RM). We assessed the fecal microbiome in fifty SM and thirty RM in total, and conducted analyses of the effect of SIV-status, species, and housing. When examining indoor-outdoor and indoor-only housing in our SM cohorts, biodiversity reduction and mild phylogenetic taxonomic perturbances were present. No statistically relevant differences were seen for biodiversity richness and evenness, or phylogenetic taxonomic communities between SIV negative and positive SM cohorts. In contrast, with pathogenic early chronic SIV infections in RM a trend of alpha diversity loss and increase of beta diversity and few phyla taxonomic communities differed. Lastly, we observed lower levels of pathobiont bacterial communities in SIV-uninfected SMs relative to RMs. These data suggest that the pre-existing bacterial community structure may contribute to the divergent phenotype between SIV natural hosts and pathogenic macaque species.\n\nImportanceHuman immunodeficiency virus remains a global concern. The sooty mangabey (SM) monkey is an important biomedical research model for understanding HIV pathogenesis due to its ability to avoid AIDS disease progression despite high viremia. In people living with HIV, gastrointestinal dysbiosis towards enrichment of pathobiont communities has been frequently reported. In this study we characterized the fecal microbiota of a primate non-pathogenic SIV host, the SM, and made direct comparisons to a pathogenic SIV host species, the rhesus macaque. We observed that SMs exhibit stability of the microbiota community into chronic SIV infection, which contrasts with SIV-infected rhesus macaques, in which we observed bacterial community divergence relative to uninfected animals. Collectively, our observation of stabilization of beneficent taxa in the mucosa of AIDS-resistant primates suggests that therapeutic strategies to enrich these communities may have potential for ameliorating the gastrointestinal inflammation in people living with HIV.

immunology↗

bioRxiv2018

In vivo measurements reveal a single 5’ intron is sufficient to increase protein expression level in C. elegans

Introns can increase gene expression levels using a variety of mechanisms collectively referred to as Intron Mediated Enhancement (IME). To date, the magnitude of IME has been quantified in human cell culture and plant models by comparing intronless reporter gene expression levels to those of intron-bearing reporter genes in vitro (mRNA, Western Blots, protein activity), using genome editing technologies that lacked full control of locus and copy number. Here, for the first time, we quantified IME in vivo, in terms of protein expression levels, using fluorescent reporter proteins expressed from a single, defined locus in Caenorhabditis elegans. To quantify the magnitude of IME, we developed a microfluidic chip-based workflow to mount and image individual animals, including software for operation and image processing. We used this workflow to systematically test the effects of position, number and sequence of introns on two different proteins, mCherry and mEGFP, driven by two different promoters, vit-2 and hsp-90. We found the three canonical synthetic introns commonly used in C. elegans transgenes increased mCherry protein concentration by approximately 50%. The naturally-occurring introns found in hsp-90 also increased mCherry expression level by about 50%. Furthermore, and consistent with prior results examining mRNA levels, protein activity or phenotypic rescue, we found that a single, natural or synthetic, 5 intron was sufficient for the full IME effect while a 3 intron was not. IME was also affected by protein coding sequence (50% for mCherry and 80% for mEGFP) but not strongly affected by promoter 46% for hsp-90 and 54% for the stronger vit-2. Our results show that IME of protein expression in C. elegans is affected by intron position and contextual coding sequence surrounding the introns, but not greatly by promoter strength. Our combined controlled transgenesis and microfluidic screening approach should facilitate screens for factors affecting IME and other intron-dependent processes.

genetics↗

bioRxiv2018

Age-dependent deterioration of nuclear pore assembly in mitotic cells decreases transport dynamics

Nuclear transport is facilitated by the Nuclear Pore Complex (NPC) and is essential for life in eukaryotes. The NPC is a long-lived and exceptionally large structure. We asked whether NPC function is compromised in ageing mitotic cells. By imaging of single yeast cells during ageing, we show that the abundance of several NPC components and NPC assembly factors decreases while signs of misassembled NPCs appear. Consequently, nuclear permeability decreases, resulting in decreased dynamics of transcription factor shuttling and increased nuclear compartmentalisation. In support that declining NPC quality control is important in mitotic ageing, we find that the transport kinetics observed in ageing is mimicked in an NPC assembly mutant. Additionally, the single cell life histories reveal that cells that better maintain NPC function are longer lived. We conclude that assembly and quality control of NPCs are major challenges for ageing mitotic cells.

cell biology↗

bioRxiv2018

Dynamics of age-related catastrophic mitotic failures and recovery in yeast

Genome instability is a hallmark of aging and contributes to age-related disorders such as progeria, cancer, and Alzheimers disease. In particular, nuclear quality control mechanisms and cell cycle checkpoints have generally been studied in young cells and animals where they function optimally, and where genomic instability is low. Here, we use single cell imaging to study the consequences of increased genomic instability during aging, and identify striking age-associated genome missegregation events. During these events the majority of mother cell chromatin, and often both spindle poles, are mistakenly sent to the daughter cell. This breakdown in mitotic fidelity is accompanied by a transient cell cycle arrest that can persist for many hours, as cells engage a retrograde transport mechanism to return chromosomes to the mother cell. The repetitive ribosomal DNA (rDNA) has been previously identified as being highly vulnerable to age-related replication stress and genomic instability, and we present several lines of evidence supporting a model whereby expansion of rDNA during aging results in nucleolar breakdown and competition for limited nucleosomes, thereby increasing risk of catastrophic genome missegregation.

molecular biology↗