Biology subjects

Oliver, J.

Publications and source records attributed to Oliver, J..

17 recordsLinked to original sources

bioRxiv2025

Canonical nuclear envelope protein emerin regulates structure and integrity of the erythrocyte plasma membrane

Red blood cells (RBC) are essential for the survival of aerobic organisms. Mutations in RBC plasma membrane proteins can give rise to a spectrum of diseases characterized by hemolysis, inefficient gas exchange, and anemia. We tested whether emerin (EMD), a nuclear envelope (NE) protein, is also expressed in anuclear cell types to regulate membrane structure and function. We find that emerin is localized to the RBC plasma membrane and regulates the localization of other membrane proteins involved in actin branching and cytoskeletal dynamics. Additionally, we find that loss of Emd in mice results in decreased RBC corpuscular volume and hemoglobin content in both sexes, as well as a male-specific increase in RBC number and hematocrit, consistent with other male-dominated EMD phenotypes. Along with decreased RBC size, we observed a reduction in the rate of osmotic lysis in Emd KO animals, particularly males. We found that human EMD mutations also have a statistically significant association with RBC phenotypes, namely MCV. Individuals with emerin-related Emery-Dreifuss muscular dystrophy (EDMD) also demonstrate a mild yet consistent RBC phenotype according to patient CBC data. Together, these data suggest that emerin can have cell-specific localization and functions independent of its canonical repertoire in nucleated cells.

cell biology↗

bioRxiv2024

Deep Quantitative Proteomics Identifies Conserved Proteome Alterations in Low and High-Grade Serous Ovarian Cancers

BackgroundLow-grade serous ovarian carcinoma (LGSOC) is a rare and largely chemoresistant subtype of epithelial ovarian cancer. Unlike treatment for high-grade serous ovarian cancer (HGSOC), management options for LGSOC patients are limited, in part, due to a lack of deep molecular characterization of this disease. To address this limitation, we aimed to define highly conserved proteome alterations in LGSOC by performing deep quantitative proteomic analysis of tumors collected from LGSOC (n=12) and HGSOC (n=24) patients or normal fallopian tube tissues (n=12) and validating proteins within two independent proteomic datasets of LGSOC (n=25) and HGSOC (n=49) tumors. ResultsOur efforts identified 275 protein alterations conserved between LGSOC and HGSOC tumors that exhibit high quantitative correlation between discovery and validation cohorts (Spearman Rho [≥] 0.82, P < 1E-4). Conserved proteins elevated in LGSOC tumors were enriched for pathways regulating cell adhesion and defective cellular apoptosis signaling and candidates mapping as putative drug targets included 5-nucleotidase/ cluster of differentiation 73 (NT5E/CD73). We also identified MUC16 (CA125) as significantly elevated in LGSOC versus HGSOC tumors and confirmed this by immunohistochemistry analysis between three independent reviewers. We also find that MUC16 exhibits a more apical versus membrane-staining pattern in LGSOC tumors, suggesting unique regulation of MUC16 in this disease subtype. ConclusionOur efforts define highly conserved protein alterations distinguishing LGSOC from HGSOC tumors, including CD73, as well as the novel identification that MUC16 is elevated and exhibits more apical staining pattern in LGSOC tumor tissues. These findings deepen our molecular understanding of LGSOC and provide unique insights into highly conserved proteome alterations in LGSOC tumors.

cancer biology↗

bioRxiv2023

Emergence of a senescent and inflammatory pulmonary CD4+ T cell population prior to lung allograft failure

Lung transplantation is a life-saving therapy for end-stage pulmonary disease, but its long-term outlook is poor due to a high incidence of chronic lung allograft dysfunction (CLAD). CLAD results from alloimmune-mediated progressive fibrosis and culminates in death or the need for re-transplantation after a median of 6 years. Existing immunosuppression fails to prevent CLAD, suggesting the existence of alloimmune pathways resistant to these drugs. Here, we used mass cytometry to identify cell populations enriched in the bronchoalveolar lavage (BAL) of patients with subsequent allograft dysfunction. We show that CD4+CD57+PD1+ T cells emerge in stable lung transplant recipients in the first year post-transplant, conferring heightened risks for CLAD and death or re-transplantation. CD4+CD57+PD1+ T cells display features of senescence and secrete inflammatory cytokines. Cellular indexing by transcriptomes and epitopes (CITE-Seq) on BAL CD4+ T cells revealed the existence of 2 oligoclonal CD57+ subsets with putative cytotoxic and follicular helper functions. Finally, we observed that CD4+CD57+PD1+ T cells are associated with lung allograft fibrosis in a mouse model and in human explanted CLAD lungs, where they localize near airway epithelium and B cells. Together, our findings reveal the existence of an inflammatory T cell population that predicts future lung allograft dysfunction and may represent a rational therapeutic target in lung transplant recipients.

immunology↗

bioRxiv2023

Tick extracellular vesicles impair epidermal homeostasis through immune-epithelial networks during hematophagy

Wound healing has been extensively studied through the lens of inflammatory disorders and cancer, but limited attention has been given to hematophagy and arthropod-borne diseases. Hematophagous ectoparasites, including ticks, subvert the wound healing response to maintain prolonged attachment and facilitate blood-feeding. Here, we unveil a strategy by which extracellular vesicles (EVs) ensure blood-feeding and arthropod survival in three medically relevant tick species. Through single cell RNA sequencing and murine genetics, we demonstrate that wildtype animals infested with EV-deficient Ixodes scapularis display a unique epidermal sub-population with a mesenchymal-like transcriptional program and an overrepresentation of pathways connected to wound healing. Furthermore, tick EVs inhibit proliferation and diminish the capacity of wound closure in keratinocytes. This occurrence was linked to phosphoinositide 3-kinase activity, keratinocyte growth factor 1 (KGF-1) and transforming growth factor {beta} (TGF-{beta}) levels. Collectively, we uncovered a strategy employed by a blood-feeding arthropod that disrupts the circuitry in cutaneous wound healing, contributing to ectoparasite fitness.

microbiology↗

bioRxiv2023

ProteoMixture: A Cell Type Deconvolution Tool for Bulk Tissue Proteomics Data

Numerous multi-omic investigations of cancer tissue have documented varying and poor pairwise transcript:protein quantitative correlations and most deconvolution tools aiming to predict cell type proportions (cell admixture) have been developed and credentialed using transcript-level data alone. To estimate cell admixture using protein abundance data, we analyzed proteome and transcriptome data generated from contrived admixtures of tumor, stroma, and immune cell models or those selectively harvested from the tissue microenvironment by laser microdissection from high grade serous ovarian cancer (HGSOC) tumors. Co-quantified transcripts and proteins performed similarly to estimate stroma and immune cell admixture in two commonly used deconvolution algorithms, ESTIMATE and ConsensusTME (r [≥] 0.63). Here we have developed and optimized protein-based signatures to estimate cell admixture proportions and benchmarked these using bulk tumor proteomics data from over 150 HGSOC patients. The optimized protein signatures supporting cell type proportion estimates from bulk tissue proteomics data are available at (https://lmdomics.org/ProteoMixture/.

bioinformatics↗

bioRxiv2023

A transmission chain linking Mycobacterium ulcerans with Aedes notoscriptus mosquitoes, possums and human Buruli ulcer cases in southeastern Australia

In temperate southeastern Australia over the past two decades there has been a marked progressive increase in human cases of Buruli ulcer, an infection of subcutaneous tissue caused by Mycobacterium ulcerans. Native possums are the major local environmental reservoir of M. ulcerans as they not only develop Buruli lesions but they also shed M. ulcerans in their excreta. However the way humans acquire M. ulcerans from possums has not been determined. Previous case-control studies, insect field surveys and vector competence studies have suggested a role for mosquitoes in M. ulcerans transmission between possums and humans. To explore these links we conducted an extensive, 4-month structured mosquito field survey and four ad hoc field surveys across an area of 350km2 on the Mornington Peninsula, an area endemic for Buruli ulcer to the south of the major metropolitan city of Melbourne. We then compared spatial and temporal patterns of M. ulcerans-positive mosquito occurrence with M. ulcerans-positive possums (established by previous possum excreta surveys) and human Buruli ulcer cases across the region. We used metabarcoding to assess mosquito blood-feeding host preference and to reconstruct M. ulcerans genomes from positive mosquitoes to test epidemiological inferences. We collected 66,325 mosquitoes spanning 26 different species from 180 repeatedly sampled traps over a 4-month period. Culex molestus and Aedes notoscriptus were the dominant species (42% and 35% of trapped mosquitoes, respectively). PCR screening 25% of trapped mosquitoes revealed a significant association between M. ulcerans and Ae. notoscriptus (p<0.0001) with a maximum likelihood estimate (MLE) of 5.88 M. ulcerans positive mosquitoes per 1,000 tested. Using spatial scanning statistics, we also observed significant overlap between clusters of M. ulcerans-positive Ae. notoscriptus, M. ulcerans-positive possum excreta and human Buruli ulcer cases. Metabarcoding analyses of blood-fed Ae. notoscriptus showed individual mosquitoes had fed both on humans and native possums. Enrichment genome sequencing from PCR-positive mosquitoes confirmed shared M. ulcerans genome single-nucleotide polymorphism (SNP) profiles between mosquitoes, possum excreta and clinical human isolates within the same regions. These findings indicate that Ae. notoscriptus likely transmit M. ulcerans in southeastern Australia and highlight mosquito control as a plausible means to control the Buruli ulcer epidemic in our region.

microbiology↗

bioRxiv2021

Perfluorooctanoic Acid Induces Liver and Serum Dyslipidemia in Humanized PPARα Mice Fed an American Diet

Per- and polyfluoroalkyl substances (PFAS) are pervasive in the environment resulting in nearly universal detection in people. Human serum PFAS concentrations are strongly associated with increased serum low-density lipoprotein cholesterol (LDL-C), and growing evidence suggests an association with serum triacylglycerides (TG). Here, we tested the hypothesis that perfluorooctanoic acid (PFOA) dysregulates liver and serum triacylglycerides in human peroxisome proliferator activated receptor (hPPAR)-expressing mice fed an American diet. Mice were exposed to PFOA (3.3 mg/l) in drinking water for 6 weeks resulting in a serum concentration of 48 {+/-} 9 g/ml. In male and female hPPAR mice, PFOA increased total liver TG and TG substituted with saturated and monounsaturated fatty acids. Lack of expression of hPPAR alone also increased total liver TG, and PFOA treatment had little effect on liver TG in null mice. In hPPAR mice, PFOA neither significantly increased nor decreased serum TG; however, there was a modest increase in TG associated with very low-density cholesterol particles in both sexes. Across studies, a non-monotonic effect of PFOA on serum TG is evident, with the serum PFOA concentration in this study falling near the "null point" between increasing and decreasing serum TG. Intriguingly, in female PPAR null mice, PFOA significantly increased serum TG, with a similar trend in males. PFOA also modified fatty acid and TG homeostasis-related gene expression in liver, in a hPPAR-dependent manner, but not in adipose. The results reveal the importance of context (serum concentration and genotype) in determining the effect of PFOA on lipid homeostasis.

pharmacology and toxicology↗

bioRxiv2021

Size and shape regional differentiation during the development of the spine in the nine-banded armadillo (Dasypus novemcinctus)

Xenarthrans (armadillos, anteaters, sloths and their extinct relatives) are unique among mammals in displaying a distinctive specialization of the posterior trunk vertebrae - supernumerary vertebral xenarthrous articulations. This study seeks to understand how xenarthry develops through ontogeny and if it may be constrained to appear within pre-existing vertebral regions. Using 3D geometric morphometrics on the neural arches of vertebrae, we explore phenotypic, allometric, and disparity patterns of the different axial morphotypes during ontogeny of nine-banded armadillos. Shape-based regionalisation analyses showed that adult thoracolumbar column is divided into three regions according to the presence or absence of ribs and the presence or absence of xenarthrous articulations. A three-region-division was retrieved in almost all specimens through development, although younger stages (e.g. foetuses, neonates) have more region boundary variability. In size-based regionalisation analyses, thoracolumbar vertebrae are separated into two regions: a pre-diaphragmatic, pre-xenarthrous region, and a post-diaphragmatic xenarthrous region. We show that posterior thoracic vertebrae grow at a slower rate, while anterior thoracics and lumbar grow at a faster rate relatively, with rates decreasing anteroposterioly in the former and increasing anteroposterioly in the latter. We propose that different proportions between vertebrae and vertebral regions might result from differences in growth pattern and timing of ossification.

developmental biology↗

bioRxiv2020

Non-opioid analgesia based on Gα signalling bias

The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, BnOCPA, is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPAs unique and exquisitely selective activation of Gob among the six Gi/o subtypes, and in the absence of {beta}-arrestin recruitment. BnOCPA thus demonstrates a highly-specific G-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective G agonism. Short summaryWe describe the selective activation of an adenosine A1 receptor-mediated intracellular pathway that provides potent analgesia in the absence of sedation or cardiorespiratory depression, paving the way for novel medicines based on the far-reaching concept of selective G agonism.

physiology↗

bioRxiv2020

Purifying selection can reduce intraspecific mitochondrial gene variation to that of slowly-evolving nuclear rRNA

Mitochondrial DNA (mtDNA) has long been used to date the divergence between species, and to explore the time when species effective population sizes changed. The idea that mitochondrial DNA is useful for molecular dating rests on the premise that its evolution is neutral. This premise was questionable to begin with, and even though it has long been challenged, the evidence against clock-like evolution of mtDNA is usually ignored. Here, we present a particularly clear and simple example to illustrate the implications of violations of the assumption of selective neutrality. DNA sequences were generated for the mtDNA COI gene and the nuclear 28S rRNA of two closely related and widely distributed rocky shore snails whose geographical ranges are defined by different thermal preferences. To our knowledge, this is the first study to use nuclear rRNA sequence for studying species-level genealogies instead of phylogenetics, presumably because this marker is considered to be uninformative at this taxonomic level. Even though the COI gene evolves at least an order of magnitude faster, which was reflected in high inter-specific divergence, intraspecific genetic variation was similar for both markers. As a result, estimates of population expansion times based on mismatch distributions were completely different for the two markers. Assuming that 28S evolves effectively clock-like, these findings likely illustrate variation-reducing purifying selection in mtDNA at the species level, and an elevated divergence rate caused by divergent selection between the two species. Although these two selective forces together make mtDNA suitable as a DNA barcoding marker because they create a barcoding gap, estimates of demographic change can be expected to be highly unreliable. Our study contributes to the growing evidence that the utility of mtDNA beyond DNA barcoding is limited.

genetics↗

bioRxiv2020

Duplications drive diversity in Bordetella pertussis on an underestimated scale.

Bacterial genetic diversity is often described using solely base pair changes despite a wide variety of other mutation types likely being major contributors. Tandem duplications of genomic loci are thought to be widespread among bacteria but due to their often intractable size and instability, comprehensive studies of the range and genome dynamics of these mutations are rare. We define a methodology to investigate duplications in bacterial genomes based on read depth of genome sequence data as a proxy for copy number. We demonstrate the approach with Bordetella pertussis, whose insertion sequence element-rich genome provides extensive scope for duplications to occur. Analysis of genome sequence data for 2430 B. pertussis isolates identified 272 putative duplications, of which 94% were located at 11 hotspot loci. We demonstrate limited phylogenetic connection for the occurrence of duplications, suggesting unstable and sporadic characteristics. Genome instability was further described in-vitro using long read sequencing via the Nanopore platform. Clonally derived laboratory cultures produced heterogenous populations containing multiple structural variants. Short read data was used to predict 272 duplications, whilst long reads generated on the Nanopore platform enabled the in-depth study of the genome dynamics of tandem duplications in B. pertussis. Our work reveals the unrecognised and dynamic genetic diversity of B. pertussis and, as the complexity of the B. pertussis genome is not unique, highlights the need for a holistic and fundamental understanding of bacterial genetics.

microbiology↗

bioRxiv2020

Perfluorooctanoic acid activates multiple nuclear receptor pathways and skews expression of genes regulating cholesterol homeostasis in liver of humanized PPARα mice fed an American diet

Humans are exposed to per- and polyfluoroalkyl substances (PFAS) in their drinking water, food, air, dust in their homes, and by direct use of consumer products. Increased concentrations of serum total cholesterol and low density lipoprotein cholesterol are among the endpoints best supported by epidemiology. The objectives of this study were to generate a new model for examining PFAS-induced dyslipidemia and to conduct molecular studies to better define mechanism(s) of action. We tested the hypothesis that PFOA exposure at a human-relevant level dysregulates expression of genes controlling cholesterol homeostasis in livers of mice expressing human PPAR (hPPAR). Female and male hPPAR and PPAR null mice were fed a diet based on the "What we eat in America" analysis and exposed to perfluorooctanoic acid (PFOA) in drinking water (8 {micro}M) for 6 weeks. This resulted in a serum PFOA concentration of 48 g/ml. PFOA increased liver mass, which was associated with histologically-evident lipid accumulation. PFOA induced PPAR and constitutive androstane receptor target gene expression in liver. Expression of genes in four pathways regulating cholesterol homeostasis were also measured. PFOA decreased expression of Hmgcr in a PPAR-dependent manner. PFOA decreased expression of Ldlr and Cyp7a1 in a PPAR-independent manner. Apob expression was not changed. Gene expression in females appeared to be more sensitive to PFOA exposure than in males. This novel study design (hPPAR mice, American diet, long term exposure) generated new insight on the effects of PFOA on cholesterol regulation in the liver and the role of hPPAR.

pharmacology and toxicology↗

bioRxiv2019

Extensive Intratumor Proteogenomic Heterogeneity Revealed by Multiregion Sampling in a High-Grade Serous Ovarian Tumor Specimen

Enriched tumor epithelium, tumor-associated stroma, and whole tissue were collected by laser microdissection from thin sections across spatially separated levels of ten primary high-grade serous ovarian tumors and analyzed using proteomics (mass spectrometry and reverse phase protein microarray) and RNA-sequencing analyses. Comparative analyses of transcript and protein abundances revealed independent clustering of enriched stroma and enriched tumor epithelium, with whole tumor tissue clustering between purified collections, driven by overall tumor purity. Comparison of historic prognostic molecular subtypes for HGSOC revealed protein and transcript expression from tumor epithelium correlated most strongly with the differentiated molecular subtype, whereas stromal proteins and transcripts most strongly correlated with mesenchymal subtype. Protein and transcript abundance in tumor epithelium and stromal collections from neighboring sections exhibited decreased correlation in samples collected just hundreds of microns apart. These data reveal substantial protein and transcript expression heterogeneity within the tumor microenvironment that directly bears on prognostic signatures and underscore the need to enrich cellular subpopulations for expression profiling.

cancer biology↗

bioRxiv2019

VE-PTP participates in vasculogenic mimicry by preventing autophagic degradation of VE-cadherin

Aberrant extra-vascular expression of VE-cadherin has been observed in metastasis associated with Vasculogenic Mimicry (VM); we have recently shown that in VM prone (VM+) tumor cells VE-cadherin is mainly in the form of pVE-cadherin in Y658 allowing an increased plasticity that potentiates VM development. As excessive VE-cadherin phosphorylation is regulated by the phosphatase VEPTP in endothelial cells in the current study we analysed its role in this aberrant phenotype in malignant tumor cells. We show that human malignant melanoma cells VM+, also express VE-PTP although at lower levels than endothelial cells. The complex VE-PTP/VE-Cadherin/p120-catenin act as a safeguard to prevent VE-cadherin degradation by autophagy. Indeed, silencing of VE-PTP results in complete degradation of VE-cadherin with the features of autophagy and increases the global p120 tyrosine phosphorylation status. In summary, we show that VE-PTP is involved in VM formation and disruption of VE-PTP/VE-Cadherin/p120 complex results in enhanced autophagy in aggressive VM+ cells.

cancer biology↗

bioRxiv2019

Hematological and Neurological Expressed 1 (HN1): a predictive and pharmacodynamic biomarker of metformin response in endometrial cancers.

Preoperative use of metformin in obese women with endometrioid endometrial cancer (EEC) reduces tumor proliferation and inhibits the mammalian target of rapamycin (mTOR) pathway, though is only effective in select cases. This study sought to identify a predictive and/or pharmacodynamic proteomic signature of metformin response to tailor its pharmacologic use. Matched pre- and post-metformin treated tumor tissues from a recently completed phase 0 window trial of metformin in EEC patients (ClinicalTrials.gov: NCT01911247), were analyzed by mass spectrometry (MS)-based proteomic and immunohistochemical analyses. Hematological and neurological expressed 1 (HN1) was significantly elevated in metformin responders (n=13) versus non-responders (n=7), which was also found to decrease in abundance in metformin responders following treatment; observations that were verified by immunohistochemical staining for HN1. Metformin response and loss of HN1 was assessed in RL95-2 and ACI-181 endometrial cancer cell lines. We further identified that silencing of HN1 abundance does not alter cellular response to metformin or basal cell proliferation, but that HN1 abundance does decrease in response to metformin treatment in RL95-2 and ACI-181 endometrial cancer cell lines. These data suggest that HN1 represents a predictive and pharmacodynamic biomarker of metformin response that, if validated in larger patient populations, may enable pre-operative EEC patient stratification to metformin treatment and the ability to monitor patient response.\n\nNovelty and ImpactHematological and Neurological Expressed 1 (HN1) is elevated in endometrioid endometrial cancer (EEC) patients that respond to metformin vs non-responders and decreases after treatment. HN1 decreases after metformin treatment of EEC cells in vitro, but is not necessary for metformin response or proliferation. We report HN1 as a novel predictive and pharmacodynamic biomarker of metformin response in EEC that may enable pre-operative EEC patient stratification to metformin treatment and the ability to monitor patient response.

cancer biology↗

bioRxiv2019

Faster Cognitive Decline in Dementia due to Alzheimer Disease with Clinically Undiagnosed Lewy Body Disease

Neuropathology has demonstrated a high rate of comorbid pathology in dementia due to Alzheimers disease (ADD). The most common major comorbidity is Lewy body disease (LBD), either as dementia with Lewy bodies (AD-DLB) or Alzheimers disease with Lewy bodies (AD-LB), the latter representing subjects with ADD and LBD not meeting neuropathological distribution and density thresholds for DLB. Although it has been established that ADD subjects with undifferentiated LBD have a more rapid cognitive decline than those with ADD alone, it is still unknown whether AD-LB subjects, who represent the majority of LBD and approximately one-third of all those with ADD, have a different clinical course. Subjects with dementia included those with \"pure\" ADD (n = 137), AD-DLB (n = 64) and AD-LB (n = 114), all with two or more complete Mini Mental State Examinations (MMSE) and a full neuropathological examination. Linear mixed models assessing MMSE change showed that the AD-LB group had significantly greater decline compared to the ADD group ({beta} = -0.69, 95% CI: -1.05, -0.33, p<0.001) while the AD-DLB group did not ({beta} = -0.30, 95% CI: -0.73, 0.14, p = 0.18). Of those with AD-DLB and AD-LB, only 66% and 2.1%, respectively, had been diagnosed with LBD at any point during their clinical course. The probable cause of LBD clinical detection failure is the lack of a sufficient set of characteristic core clinical features. Core DLB clinical features were not more common in AD-LB as compared to ADD. Compared with clinically-diagnosed AD-DLB subjects, those that were clinically undetected had significantly lower prevalences of parkinsonism (p = 0.046), visual hallucinations (p = 0.0008) and dream enactment behavior (0.013). Clinical identification of ADD with LBD would allow stratified analyses of ADD clinical trials, potentially improving the probability of trial success.

neuroscience↗

bioRxiv2017

Rational Design of Evolutionarily Stable Microbial Kill Switches

The evolutionary stability of synthetic genetic circuits is key to both the understanding and application of genetic control elements. One particularly useful but challenging situation is a switch between life and death depending on environment. Here are presented \"essentializer\" and \"cryodeath\" circuits, which act as kill switches in Escherichia coli. The essentializer element induces cell death upon the loss of a bi-stable cI/Cro memory switch. Cryodeath makes use of a cold-inducible promoter to express a toxin. We employ rational design and a novel toxin/antitoxin titering approach to produce and screen a small library of potential constructs, in order to select for constructs that are evolutionarily stable. Both kill switches were shown to maintain functionality in vitro for at least 140 generations. In addition, cryodeath was shown to control the growth environment of a bacterial population, with an escape rate of less than 1 in 105 after ten days in vivo.

synthetic biology↗