Biology subjects

Mitchell, G.

Publications and source records attributed to Mitchell, G..

13 recordsLinked to original sources

bioRxiv2025

Screening of a chemogenetic library unveils dual-stage antimalarials with low cytotoxicity

The global effort to eradicate malaria requires the development of drugs with novel mechanisms of action and differentiated pharmacological profiles. Both liver and blood phases of Plasmodium infection are essential for disease progression and transmission, making them primary targets for therapeutic intervention. In this study, we established a dual-luciferase reporter assay to simultaneously assess Plasmodium berghei liver-stage development and host cell viability. Screening a library of bioactive small molecules with annotated human targets revealed several inhibitors with potent liver-stage activity and minimal cytotoxicity. Further profiling against P. falciparum asexual blood stages identified compounds with dual-stage activity, underscoring their potential utility for development as either prophylactic or therapeutic agents. Knowledge of their human targets will facilitate investigation into host- or parasite- directed antimalarial mechanisms, thereby accelerating both biological insight and rational drug development. Our findings constitute a valuable resource for research on malaria biology and the discovery of next-generation antimalarials.

microbiology↗

bioRxiv2025

NTBC dosing and outcomes in hereditary tyrosinemia type 1: insights from a representative human model and 99 patients

Hereditary tyrosinemia type 1 (HT1) is a rare and severe metabolic liver disorder caused by fumarylacetoacetate hydrolase (FAH) deficiency. The optimal dose and long-term effects of the only available treatment, nitisinone (NTBC), remain unclear due to the absence of clinical trial data. Here, we generated a representative human in vitro model of HT1 using iPSC-derived hepatocytes, which faithfully recapitulated key disease features. We investigated the mechanisms of FAH deficiency-induced hepatocellular injury and evaluated the effects of NTBC treatment. We confirmed treatment efficacy and identified 50 {micro}mol/L as the minimal effective NTBC concentration to prevent cellular damage. This protective dose was subsequently validated in a large cohort of 99 HT1 patients, providing compelling evidence for establishing minimal therapeutic NTBC levels. Notably, approximately 10% of disease-associated genes, many implicated in hepatocellular carcinoma, remained dysregulated despite treatment, raising concerns that NTBC may not fully eliminate long-term oncogenic risk.

genetics↗

bioRxiv2025

Neutrophil extracellular traps offer a new therapeutic target for elephant endotheliotropic herpes hemorrhagic disease (EEHV-HD)

Elephant survival is threatened by a devastating hemorrhagic disease called elephant endotheliotropic herpes virus-hemorrhagic disease (EEHV-HD). Once clinical signs are observed in elephants, the disease progresses rapidly and frequently results in death. EEHV-HD negatively impacts elephant conservation because very young, reproductively immature elephants are most at risk for death. Ongoing efforts to understand disease pathogenesis and progression may identify treatment targets and improve clinical outcomes. In some lethal EEHV-HD cases, microthrombosis has been observed in organ tissues similar to other hemorrhagic diseases in humans and animals where sticky webs of protein-coated DNA strands called neutrophil extracellular traps (NETs) exacerbate thrombosis and hemorrhage associated with disseminated intravascular coagulation (DIC). In this study, we sought to identify if NET formation occurs in elephants and could contribute to poor outcomes in EEHV-HD. Our study demonstrated NET release for the first time from elephant heterophils (neutrophils) that occurred in response to various stimuli, including plasma from EEHV-HD affected elephants. EEHV-HD affected tissues contained extensive NETs suggesting that dysregulated NET formation contributes to pathogenesis of this disease. Importantly, elephant neutrophils were blocked from releasing NETs in response to EEHV-HD plasma using known NET inhibitors. The ability to stop NETs in EEHV-HD offers a new therapeutic approach that could be combined with current therapies to improve survival for affected elephants and to positively impact conservation efforts.

immunology↗

bioRxiv2025

An unusual genetic switch controls Mycobacterium avium pathogenesis, antibiotic resistance and colony morphology

Mycobacterium avium subspecies hominissuis (Mah) is an emerging environmental pathogen highly adapted to a wide range of niches, from treated water systems to mammalian tissues. On solid media, Mah forms two distinct colony morphologies, smooth transparent (SmT) and smooth opaque (SmO). These colony morphologies are representative of broader differential phenotypic states in which SmT cells are virulent and have high resistance to antibiotics while SmO cells are avirulent, antibiotic-sensitive and grow faster than SmT cells in culture. Importantly, Mah interconverts between these two morphotypes but the mechanism of SmT-SmO switching is unknown. Here we show that SmT-SmO switching is governed by a reversible transposition event that regulates expression of a periplasmic lipoprotein, Erp (extracellular repetitive protein). We found that transposition of IS1245, an endogenous insertion sequence, into the erp gene correlated with the SmT-SmO transition, and its precise removal coincided with the switch back to SmT. Genetic analyses showed that erp is required for maintenance of the SmT state and sufficient to drive the switch from SmO to SmT. We also identified a mutation in a periplasmic protease, MarP, that locks Mah in the SmO state and blocks erp-mediated switching to SmT. Our results indicate that Erp and MarP function in a signal transduction pathway that regulates a broad transcriptional response to periplasmic stress. Moreover, identification of components that control Mah colony morphology switching has revealed a potential new strategy for combating the inherent antibiotic resistance of Mycobacterium avium infections.

microbiology↗

bioRxiv2024

Tax1bp1 enhances bacterial virulence and promotes inflammatory responses during Mycobacterium tuberculosis infection of alveolar macrophages

Crosstalk between autophagy, host cell death, and inflammatory host responses to bacterial pathogens enables effective innate immune responses that limit bacterial growth while minimizing coincidental host damage. Mycobacterium tuberculosis (Mtb) thwarts innate immune defense mechanisms in alveolar macrophages (AMs) during the initial stages of infection and in recruited bone marrow-derived cells during later stages of infection. However, how protective inflammatory responses are achieved during Mtb infection and the variation of the response in different macrophage subtypes remain obscure. Here, we show that the autophagy receptor Tax1bp1 plays a critical role in enhancing inflammatory cytokine production and increasing the susceptibility of mice to Mtb infection. Surprisingly, although Tax1bp1 restricts Mtb growth during infection of bone marrow-derived macrophages (BMDMs) (Budzik et al. 2020) and terminates cytokine production in response to cytokine stimulation or viral infection, Tax1bp1 instead promotes Mtb growth in AMs, neutrophils, and a subset of recruited monocyte-derived cells from the bone marrow. Tax1bp1 also leads to increases in bacterial growth and inflammatory responses during infection of mice with Listeria monocytogenes, an intracellular pathogen that is not effectively targeted to canonical autophagy. In Mtb-infected AMs but not BMDMs, Tax1bp1 enhances necrotic-like cell death early after infection, reprogramming the mode of host cell death to favor Mtb replication in AMs. Tax1bp1s impact on host cell death is a mechanism that explains Tax1bp1s cell type-specific role in the control of Mtb growth. Similar to Tax1bp1-deficiency in AMs, the expression of phosphosite-deficient Tax1bp1 restricts Mtb growth. Together, these results show that Tax1bp1 plays a crucial role in linking the regulation of autophagy, cell death, and pro-inflammatory host responses and enhancing susceptibility to bacterial infection. Author SummaryAlthough macrophages are the first innate immune cells to encounter Mycobacterium tuberculosis during infection, M. tuberculosis has evolved the ability to persist in them. Recent studies highlight that some types of macrophages are more permissive to M. tuberculosis replication and survival than others, but the mechanisms for cell type-specific differences in M. tuberculosis growth are only beginning to be understood. We found that the host factor, Tax1bp1 (Tax-1 binding protein 1), supports M. tuberculosis growth during animal infection and in specific subsets of innate immune cells, including alveolar macrophages while restricting M. tuberculosis in bone marrow-derived macrophages. We also found that Tax1bp1 has a similar phenotype in enhancing the pathogenesis of another intracellular pathogen, Listeria monocytogenes. Compared to bone marrow-derived macrophages, in alveolar macrophages, Tax1bp1 enhances the release of inflammatory mediators and leads to necrotic-like host cell death, which is known to enhance M. tuberculosis growth. Phosphorylation of Tax1bp1 in alveolar macrophages promotes M. tuberculosis growth. Our research highlights that Tax1bp1 is a host target for host-directed therapy against M. tuberculosis and controls host responses to M. tuberculosis in a cell type-specific manner.

microbiology↗

bioRxiv2024

Development of a p62 biodegrader for autophagy targeted degradation

Harnessing autophagy for targeted degradation is a promising extension to proteasome-based targeted protein degradation because of the capacity and versatility of lysosomes to degrade large and complex cargo, thus broadening the scope of therapeutic targets. While small-molecule degraders recruiting the autophagy machinery to targets are starting to emerge, it remains unclear which component of the autophagy lysosomal pathway is most efficacious to induce selective target degradation. Here, we describe two orthogonal induced-proximity strategies to identify and prioritize autophagy effectors that are sufficient to degrade organelles as well as soluble targets. We show that induced proximity of different effectors such as autophagy cargo receptors, ATG8-like proteins or the kinases ULK1 and TBK1 are sufficient to trigger mitophagy. In contrast, self-oligomerizing autophagy cargo receptors outperform ATG8- like effectors and autophagy-related kinases in clearing a soluble cytosolic protein. We further explore the importance of avidity for targeted degradation via autophagy and reveal that the PB1 domain of p62 fused to a LIR peptide is a minimal degron to induce the degradation of mitochondria as well as cytosolic proteins. By developing a novel and highly selective intrabody against the autophagy cargo receptor p62 into a heterobifunctional degrader, we demonstrate that recruitment of endogenous p62 is sufficient to clear mitochondria. This biodegrader, however, is unable to induce degradation of soluble cytosolic proteins due to its inhibitory effect on p62 self-oligomerization. Our study highlights the importance of avidity and suggests that autophagy cargo receptors are attractive entry points for the development of heterobifunctional degraders for complex targets such as organelles or protein aggregates.

synthetic biology↗

bioRxiv2024

Hierarchically Vascularized and Implantable Tissue Constructs created through Angiogenesis from Tissue-Engineered Vascular Grafts

A major roadblock in implementing engineered tissues clinically lies in their limited vascularization. After implantation, such tissues do not integrate with the hosts circulation as quickly as needed, commonly resulting in loss of viability and functionality. This study presents a solution to the vascularization problem that could enable the survival and function of large, transplantable, and vascularized engineered tissues. The technique allows vascularization of a cell laden hydrogel through angiogenesis from a suturable tissue-engineered vascular graft (TEVG) constructed from electrospun polycaprolactone with macropores. The graft is surrounded by a layer of cell-laden gelatin-methacryloyl hydrogel. The constructs are suturable and possess mechanical properties like native vessels. Angiogenesis occurs through the pores in the graft, resulting in a hydrogel tcontaining an extensive vascular network that is connected to an implantable TEVG. The size of the engineered tissue and the degree of vascularization can be increased by adding multiple TEVGs into a single construct. The engineered tissue has the potential to be immediately perfused by the patients blood upon surgical anastomosis to host vessels, enabling survival of implanted cells. These findings provide a meaningful step to address the longstanding problem of fabricating suturable pre-vascularized tissues which could survive upon implantation in vivo.

bioengineering↗

bioRxiv2023

Bioenergetic mapping of 'healthy microbiomes' via compound processing potential imprinted in gut and soil metagenomes

Despite mounting evidence of their importance in human health and ecosystem functioning, the definition and measurement of healthy microbiomes remain unclear. More advanced knowledge exists on health associations for compounds used or produced by microbes. Environmental microbiome exposures (especially via soils) also help shape, and may supplement, the functional capacity of human microbiomes. Given the synchronous interaction between microbes, their feedstocks, and micro-environments, with functional genes facilitating chemical transformations, there exists an intriguing opportunity to examine microbiomes in terms of their capacity to process compounds relevant to human health. Here we integrate functional genomics and biochemistry frameworks to derive new quantitative measures of the potential for human gut and environmental soil metagenomes to process major compound classes (e.g., lipids, carbohydrates) and selected biomolecules (e.g., vitamins, short-chain fatty acids) linked to human health. Metagenome functional potential profile data were translated into a universal compound mapping landscape based on bioenergetic van Krevelen mapping of function-level meta-compounds and corresponding functional relative abundances, reflecting imprinted genetic capacity of microbiomes to metabolize an array of different compounds. We show that measures of compound processing potential associated with human health and disease (examining atherosclerotic cardiovascular disease, colorectal cancer, type 2 diabetes and anxious-depressive behavior case studies), and displayed seemingly predictable shifts along gradients of ecological disturbance in plant-soil ecosystems (three case studies). Ecosystem quality explained 60-92% of variation in soil metagenome compound processing potential measures in a post-mining restoration case study dataset. With growing knowledge of the varying proficiency of environmental microbiota to process human health associated compounds, we might design environmental interventions or nature prescriptions to modulate our exposures, thereby advancing microbiota-oriented approaches to human health. Compound processing potential offers a simplified, integrative approach for applying metagenomics in ongoing efforts to understand and quantify the role and linkages of microbiota in environmental- and human-health.

bioinformatics↗

bioRxiv2022

Transcriptional profiling of hepatocytes infected with the replicative form of the malaria parasite Plasmodium cynomolgi

The zoonotic simian parasite Plasmodium cynomolgi develops into replicating schizonts and dormant hypnozoites during the infection of hepatocytes and is used as a model organism to study relapsing malaria. We previously reported the transcriptional profiling of P. cynomolgi liver stages and revealed many important biological features of the parasite (Bertschi et al., Elife, 2018; Voorberg-van der Wel et al., Elife, 2017) but left out the host response to malaria infection. Here, we used our published RNA sequencing data to quantify the expression of host genes in rhesus macaque hepatocytes infected with P. cynomolgi in comparison to either cells from uninfected samples or uninfected bystander cells. Although the dataset could not be used to resolve the transcriptional profile of hypnozoite-infected hepatocytes, it provided a snapshot of the host response to liver stage schizonts and identified specific host pathways that are modulated during malaria infection. This study constitutes a valuable resource characterizing the hepatocyte response to P. cynomolgi infection and provides a framework to build on future research that aims at understanding hepatocyte-parasite interactions during relapsing malaria infection.

microbiology↗

bioRxiv2022

Habitat protection and restoration: win-win opportunities for migratory birds in the Northern Andes

Identifying strategies that offer co-benefits for biodiversity protection, forest restoration and human well-being are important for successful conservation outcomes. In this study, we identified opportunities where forest restoration and rehabilitation programs in Colombia also align with priority areas for the conservation of Neotropical migratory birds. We used citizen science eBird-based abundance estimates to define regions with the highest richness of Neotropical migratory birds of conservation concern at montane elevations in Colombia and aligned these high richness areas with domestic initiatives for forest protection (Forest Areas), restoration (Restoration Areas) and rehabilitation (Rehabilitation Areas). We quantified the location and amounts of these three areas as well as the type of land protection and designation within them, specifically, National Protected Areas, Indigenous Reserves, Afro-descendent territories, and regions affected by poverty and violence that are prioritized for rural development by the Colombian government in Post-conflict Territorially Focused Development Programs (PDET). Almost half of Forest Areas overlapped with PDETs where goals for economic development present a risk of forest loss if not done sustainably. There was a 20% overlap between Forest Areas and Afro-descendant territories and indigenous reserves; most of this overlap was outside of established protected areas thus presenting an opportunity for community forest conservation that benefits migratory birds. We found an alignment of less than 6% between migrant bird focal areas and the priority Restoration and Rehabilitation Planning Areas identified by the Colombian National Restoration Plan indicating less opportunity for these programs to simultaneously benefit Neotropical migrant species. Our approach highlights that timely and efficient conservation of declining migrants depends on identifying the regions and strategies that incorporate local communities as part of the solution to forest loss and degradation in Colombia. HighlightsColombia covers over half of key wintering areas for migratory birds in South America Most of the migrants overwinter range overlaps with working landscapes Priority national restoration/rehabilitation areas are ineffective to benefit migrants Forest conservation needs actions involving vulnerable and minority groups

ecology↗

bioRxiv2020

Ceragenins and antimicrobial peptides kill bacteria through distinct mechanisms

Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally-occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here we report the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA131. Using transcriptomic and proteomic analyses, we found that Escherichia coli responds similarly to both CAMPs and ceragenins by inducing a Cpx envelope stress response. However, whereas E. coli exposed to CAMPs increased expression of genes involved in colanic acid biosynthesis, bacteria exposed to ceragenins specifically modulated functions related to phosphate transport, indicating distinct mechanisms of action between these two classes of molecules. Although traditional genetic approaches failed to identify genes that confer high-level resistance to ceragenins, using a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) approach we identified E. coli essential genes that when knocked down modify sensitivity to these molecules. Comparison of the essential gene-antibiotic interactions for each of the CAMPs and ceragenins identified both overlapping and distinct dependencies for their antimicrobial activities. Overall, this study indicates that while some bacterial responses to ceragenins overlap with those induced by naturally-occurring CAMPs, these synthetic molecules target the bacterial envelope using a distinctive mode of action. IMPORTANCEThe development of novel antibiotics is essential since the current arsenal of antimicrobials will soon be ineffective due to the widespread occurrence of antibiotic resistance. Development of naturally-occurring cationic antimicrobial peptides (CAMPs) for therapeutics to combat antibiotic resistance has been hampered by high production costs and protease sensitivity, among other factors. The ceragenins are a family of synthetic CAMP mimics that kill a broad spectrum of bacterial species but are less expensive to produce, resistant to proteolytic degradation and have been associated with low levels of resistance. Determining how ceragenins function may identify new essential biological pathways of bacteria that are less prone to development of resistance and will further our understanding of the design principles for maximizing the effects of synthetic CAMPs.

microbiology↗

bioRxiv2019

Estimating relative CWD susceptibility and disease progression in farmed whitetail deer with rare PRNP alleles

Chronic wasting disease is a prion disease affecting both free-ranging and farmed cervids in North America and Scandinavia. A range of cervid species have been found to be susceptible, each with variations in the gene for the normal prion protein, PRNP, reportedly influencing both disease susceptibility and progression in the respective hosts. Despite the finding of several different PRNP alleles in whitetail deer, the majority of past research has focused on two of the more common alleles identified - the 96G and 96S alleles. In the present study, we evaluate both infection status and disease stage in nearly 2100 farmed deer depopulated in the United States and Canada, including 714 CWD-positive deer and correlate our findings with PRNP genotype, including the more rare 95H, 116G, and 226K alleles. We found significant differences in either likelihood of being found infected or disease stage (and in many cases both) at the time of depopulation in all genotypes present, relative to the most common 96GG genotype. Despite high prevalence in many of the herds examined, infection was not found in several of the reported genotypes. These findings suggest that additional research is necessary to more properly define the role that these genotypes may play in managing CWD in both farmed and free-ranging whitetail deer, with consideration for factors including relative fitness levels, incubation periods, and the kinetics of shedding in animals with these rare genotypes.

microbiology↗

bioRxiv2018

Genome-wide analysis of cell cycle-regulating genes in the symbiotic dinoflagellate Symbiodinium minutum.

A critical relationship exists between reef-building corals and their photosynthetic endosymbionts. As important as this relationship is for reef health, it is exquisitely delicate--exposure to temperatures only marginally above the average summer maximum can cause corals to bleach, expelling their resident algae. Interestingly, several studies indicate that failure of corals to properly regulate symbiont cell divisions at high temperatures may cause bleaching. This needs to be further investigated, but first, it is necessary to decipher the molecular mechanisms controlingl the cell division cycle in these organisms. As a first step toward this goal, we identified key cell cycle-regulating genes in the recently published genome of the symbiotic dinoflagellate Symbiodinium minutum. We then correlated expression of these genes with cell cycle phase in diurnally growing S. minutum in culture. Of particular interest, this approach allowed us to identify cyclins and cyclin-dependent kinases that are involved in the G1/S transition--a likely point for coral cells to exert control over algal cell divisions.

genomics↗