Biology subjects

Clark, J. R.

Publications and source records attributed to Clark, J. R..

13 recordsLinked to original sources

bioRxiv2026

Lipoprotein(a) promotes thrombosis through platelet activation and promotion of a lysis-resistant thrombus architecture

Elevated levels of lipoprotein(a) (Lp(a)) are an independent risk factor for the development of atherothrombotic diseases. However, it is unknown if Lp(a) directly promotes thrombus formation, inhibits thrombus clearance, or merely accelerates the underlying atherosclerotic processes that culminate in plaque rupture. While numerous studies indicate that the apolipoprotein(a) (apo(a)) component of Lp(a) can inhibit plasminogen activation and fibrinolysis, recent evidence suggests that these effects may not be retained in Lp(a). An alternative mechanism through which Lp(a) may promote atherothrombotic events is by impacting platelet function. However, the effects of Lp(a) on platelet function and thrombosis have never been directly assessed in blood clots formed from flowing whole blood. Using a transgenic mouse model expressing high plasma concentrations of apo(a), we showed using a laser-induced mesenteric vessel injury model employing intravital microscopy that apo(a) increased platelet and fibrin volumes in the thrombi without affecting fibrinolysis. In a ferric chloride-induced mouse carotid artery thrombosis model, we found that apo(a) substantially reduced occlusion times and led to more stable thrombi; importantly, we also demonstrated that the effects of Lp(a) could be mitigated by low-dose aspirin therapy. We evaluated the prothrombotic potential of Lp(a) in human blood clots formed under arterial flow conditions using a Chandler loop apparatus. In these studies, we showed that the presence of Lp(a) during thrombogenesis inhibited lysis of the thrombi, without directly impacting fibrinolysis. Lp(a) promoted platelet accumulation in the Chandler thrombi and facilitated the development of fibrin networks that displayed features of fibrinolysis resistance. In an analysis of the UK Biobank, participants with Lp(a) [≥]125 nmol/L had a higher risk for arterial thrombosis of non-atherosclerotic etiology but not a higher risk for venous thromboembolism. Collectively, these findings demonstrate that Lp(a) is inherently prothrombotic and likely promotes arterial thrombosis in vivo in part through promoting platelet activation. These findings explain how elevated Lp(a) is an important risk factor for arterial thrombosis either with or without an atherosclerotic etiology as well as observational primary prevention data suggesting that aspirin reduces atherothrombotic risk specifically in patients with elevated Lp(a).

biochemistry↗

bioRxiv2025

A Single Genomic Region Controls Primocane Fruiting in Tetraploid Blackberry

The fresh-market blackberry (Rubus subgenus Rubus) industry has expanded dramatically in the past two decades, driven in part by improved cultivars. Introgression of the primocane-fruiting (PF; annual flowering) trait into elite germplasm has enabled dual cropping in a single year, season extension, and cultivation in tropical and subtropical regions. Despite its economic performance, the genetic basis of PF is not well understood. It has been proposed that the PF trait is controlled by a major recessive locus, but its genomic location is unclear. Here, a genome-wide association study (GWAS) of 365 tetraploid blackberry genotypes identified a single genomic region on chromosome Ra03 ([~]33 Mb) strongly associated with PF. Genetic linkage analysis in a biparental population confirmed that the same interval (32-35 Mb) was linked to the PF phenotype. Ten putative candidate genes were identified in this region. Allele mining using whole-genome resequencing of 17 genotypes highlighted two high-priority candidates: a CCCH-type zinc finger gene and a ubiquitin-specific protease gene. Use of an improved Rubus argutus Hillquist genome annotation (v1.2) enabled refined variant interpretation, including identification of regulatory 3' UTR polymorphisms in the zinc finger homolog. Two diagnostic KASP markers (PF1 and PF2), designed from the most significant GWAS SNPs, predicted the PF phenotype with over 96% accuracy in a validation panel of 494 tetraploid blackberries from multiple breeding programs. Together, these results provide the first high-resolution mapping of the PF locus in blackberry, identify candidate genes for flowering regulation in Rubus, and deliver diagnostic markers that can be immediately deployed in breeding programs.

genetics↗

bioRxiv2025

Cytokine and Immunoglobulin Dynamics in Phage Therapy: Insights from Clinical Cases

BackgroundBacteriophage therapy is increasingly used for antibiotic-refractory infections, yet its immunologic effects in humans remain poorly defined. We evaluated cytokine and immunoglobulin responses in nine compassionate-use cases of intravenous phage therapy, including solid organ transplant recipients and patients with device-associated or refractory bacterial infections. MethodsSerial serum samples were analyzed by multiplex Luminex immunology and isotyping assays, with results correlated to host immune status, infecting organism, and clinical outcome. ResultsCytokine patterns were heterogeneous and appeared to reflect the infecting pathogen more than phage exposure. Pseudomonas aeruginosa infections elicited broad pro-inflammatory cytokine increases (notably MCP-3 and TNF-{beta}), while Staphylococcus aureus infection was associated with overall cytokine reduction except for IL-6. Most immunocompetent patients exhibited an early IgM response within 1-2 weeks of phage initiation, whereas immunocompromised hosts demonstrated attenuated antibody levels. Neither cytokine nor humoral responses correlated with clinical outcome. In one case, serum neutralization developed against a specific phage but not against a subsequent, distinct phage cocktail targeting the same organism, suggesting variability in phage immunogenicity. ConclusionOverall, intravenous phage therapy frequently induces IgM responses, which are more pronounced in immunocompetent patients, while cytokine dynamics depend more on pathogen and immune status than on phage administration itself. These findings underscore the need for standardized immune profiling in clinical phage trials to delineate beneficial versus detrimental immune responses and to inform the design of phage formulations with optimized immunogenic profiles.

microbiology↗

bioRxiv2025

Strainify: Strain-Level Microbiome Profiling for Low-Coverage Short-Read Metagenomic Datasets

MotivationStrain-level microbiome profiling has revealed key insights into microbial community composition and strain dynamics. However, accurate strain-level analysis remains challenging due to limited linkage information, ambiguous read mapping, and complicating factors such as genome similarity, sequencing depth, and community complexity. These challenges are especially pronounced for short-read metagenomic data when estimating the relative abundances of multiple strains, a task critical for genotype-phenotype association studies. ResultsTo address this gap, we present Strainify, which enables accurate strain-level abundance estimation from short-read metagenomes with as little as 1% genome coverage. Specifically, Strainify combines (1) identification of informative variants via core genome alignment, (2) filtering of confounding variants via a window-based test, and (3) maximum likelihood estimation of strain abundances. A Shannon entropy-weighted version of the model further improves robustness in noisy, low-coverage settings by downweighting sites with low information content. Across simulated communities of varying complexity, Strainify consistently outperformed existing approaches. On mock community sequencing data, Strainifys estimates aligned more closely with reference abundances. When applied to a longitudinal gut microbiome dataset, Strainify successfully recapitulated the reported temporal dynamics of Bacteroides ovatus strain groups, demonstrating its ability to recover biologically meaningful patterns from real-world metagenomes. Together, these results establish Strainify as a robust and versatile solution for accurate strain-level abundance estimation in short-read, low-coverage microbiome studies. AvailabilityThe Strainify code and results are available at: https://github.com/treangenlab/Strainify

bioinformatics↗

bioRxiv2025

Pathogen-Phage Geomapping to Overcome Resistance

The rise of antibiotic resistance has renewed interest in bacteriophages as therapeutic alternatives. However, co-evolution of phage and bacteria will naturally give rise to phage-resistant pathogens, complicating phage therapy efforts. A critical bottleneck in the production of phage therapeutics is the discovery of virulent phages against resistant pathogens. Conventional methods for discovery are time-consuming, biased, and laborious, limiting the potential for identifying suitable phage candidates. To overcome these limitations, we combined small-volume environmental sampling with 16S rRNA sequencing to identify reservoirs where bacterial hosts co-exist with their phage predators. This strategy, which we term geographical phage mapping (ge{Phi}mapping), pinpoints ecological "hotspots" for targeted phage hunting. We further developed a portable phage hunting device ({Phi}HD) that generates highly enriched phage concentrates directly from these reservoirs. By integrating ge{Phi}mapping with high-throughput enrichment, we constructed the R{Phi} library, a diverse collection of novel phages. We captured and isolated 36 new phages targeting extremely-resistant organisms across various ESKAPE pathogens when conventional phage hunting and experimental evolution approaches failed.

microbiology↗

bioRxiv2024

Genetic Control of Prickles in Tetraploid Blackberry

Prickle-free blackberry (Rubus subgenus Rubus) canes are strongly preferred by growers due to food and worker safety concerns and damage to fruit from mechanical injury by prickles. This project was conducted to identify the genetic region responsible for prickle-free canes derived from the recessive Merton Thornless source in autotetraploid blackberry using a genome-wide association study, develop diagnostic KASP markers for prickle-free canes, and determine the effects of allele dosage at the prickle-free locus on prickle density in two biparental populations. The prickle locus was located on chromosome Ra04 from 30.48 to 36.04 Mb in an extensive LD block, with the peak SNP located at 33.64 Mb. Two diagnostic KASP markers were developed that correctly predicted the phenotype of 97% and 96% of 626 diverse fresh-market blackberry genotypes from multiple breeding programs, respectively. Allele dosage at the prickle-free locus had a significant impact on prickle density, with duplex prickly genotypes having significantly higher prickle density than simplex genotypes in both biparental populations studied. Five potential candidate genes with functional annotations related to epidermal, trichome, and/or prickle development were identified within the prickle-free locus, but no nonsynonymous polymorphism within these genes were identified. Article SummaryBlackberry cultivars with prickle-free canes are strongly preferred by growers and shipper marketers. The objectives of this study were to map the prickle-free locus in tetraploid fresh- market blackberries using a genome-wide association approach and develop diagnostic molecular markers that breeders can use to plan crosses and screen seedlings for prickle-free canes. The prickle-free locus was mapped to a 5.6 Mb region on chromosome Ra04. Two molecular markers developed in this region correctly predicted the phenotype of 96% or more of the breeding selections and cultivars in a large validation panel composed of germplasm from three breeding programs.

genetics↗

bioRxiv2024

Eco-Microbiology: Discovering Biochemical Enhancers of PET Biodegradation by Piscinibacter sakaiensis

The scale of plastic pollution boggles the mind. Nearly 400 megatons of virgin plastics are produced annually, with an environmental release rate of 80 percent; and plastic waste including micro- and nanoplastics are associated with a plethora of problems. The naturally evolved abilities of plastic-degrading and consuming microbes offer a starting point for generating sustainable and eco-centric solutions to plastic pollution. Here we developed an iterative discovery procedure coupling faster quasi-high-throughput polyethylene terephthalate (PET) dependent bioactivity screens with longer-term PET biodegradation assays to find small molecule and ionic boosters of PET consumption by the bacterium Piscinibacter sakaiensis. We discovered multiple hits supporting greater than 2-fold enhancement of PET biodegradation - with hits belonging to a small but heterogeneous set of compounds and mixtures, suggesting upregulation of PET consumption via multiple paths. This work has the potential to advance the creation of a fermentation-based process for solving PET plastic pollution. ImportancePlastic pollution is an urgent environmental issue. In addition, micro- and nanoplastics (MNPs) have become an acute source of worry with discoveries of the global distribution and transport of MNPs, their presence within a diversity of organisms including common foodstuffs and human tissues, and their potential association with declining fertility and various disease states. Solutions are needed and the microbial world offers abundant help via naturally evolved biodegraders of plastic waste. We created a non-genetic method to accelerate polyethylene terephthalate (PET) plastic biodegradation by Piscinibacter sakaiensis, a bacterium that evolved to slowly but completely consume PET. Our method entails a combination of plastic-dependent bioactivity screens and slower biodegradation tests to find extrinsic biochemical stimulators of PET biodegradation. The conditions we found boost PET biodegradation by over two-fold and provide a foundation for further studies to realize a fermentation-based process needed to solve PET plastic pollution.

microbiology↗

bioRxiv2024

Spatial mapping reveals unique cellular interactions and enhanced tertiary lymphoid structures in responders to anti-PD-1 therapy in mucosal head and neck cancers.

Survival in recurrent/metastatic head and neck mucosal squamous cell carcinoma (HNmSCC) remains poor. Anti-programmed death (PD)-1 therapies have demonstrated improved survival with lower toxicity when compared to standard chemotherapy. However, response to anti-PD-1 therapy remains modest, at 13-17%. We evaluated the tumor microenvironment (TME) using Imaging Mass Cytometry (IMC) on 27 tumor specimens from 24 advanced HNmSCC patients prior to receiving anti-PD-1 based treatment. We show significantly increased central memory T cells and B cells in responders (n=8) when compared to non-responders (n=16). Spatial mapping identified interactions between phenotypically distinct malignant squamous cells with CD8+ T cells, CD4+ Tregs and endothelial cells in responders, and avoidance of these cells in non-responders. Importantly, regional analysis shows responders have more abundant tertiary lymphoid structures (TLS), with TLS proportion >20% also associated with longer progression free survival. Together these findings define the immune landscape associated with response to anti-PD-1 treatment in HNmSCCs.

cancer biology↗

bioRxiv2023

Progress toward the development of an effective vaccine for Extraintestinal pathogenic E. coli (ExPEC): The application of the multiple-protein subunits vaccine in different murine models

Extraintestinal pathogenic E. coli (ExPEC) is the primary Gram-negative bacterial pathogen, and the leading cause of life-threatening sepsis and urinary tract infections (UTI) in adults. The emergence and increasing prevalence of multidrug-resistance (MDR) ExPEC strains have led to considerable treatment failures, increased hospitalization rates, morbidity, and mortality. A prophylactic vaccine against ExPEC has the potential to reduce severe infection-related morbidity and mortality, helping to address the escalating antimicrobial resistance (AMR) crisis worldwide. The -hemolysin (HlyA) is a critical, frequently detected secreted cytotoxic virulence factor in ExPEC, with HlyA-expressing ExPEC strains correlating with increased severity and infection dissemination in clinical levels. In this study, we assessed the protective efficacy of pro-HlyA (the inactive and immature precursor of HlyA) and Dual-Hit (a combination of pro-HlyA and SinH-3, the previously reported immunoglobulin-like domain-3 of the invasin-like autotransporter protein SinH), as ExPEC vaccine candidates. We demonstrated that immunizing mice with pro-HlyA or Dual-Hit significantly reduced bacterial burden and increased survival rates against pandemic ExPEC sequence type strains, ST73 (CFT073) and ST95 (UTI89), in the model of bacteremia and mortality. Both pro-HlyA or Dual-Hit immunizations also provided significant protection against UTI89 colonization in the bladder in the murine UTI model. Furthermore, vaccination with Dual-Hit provided enduring and robust protection against a mixture of ten typical high-virulent sequence types of ExPEC strains, resulting in a promising broad-spectrum vaccine candidate. These findings suggest that pro-HlyA and Dual-Hit might serve as highly effective vaccine targets and highlight the potential of these vaccine candidates for further development and evaluation.

microbiology↗

bioRxiv2021

Phage resistance accompanies reduced fitness of uropathogenic E. coli in the urinary environment

Urinary tract infections (UTIs) are among the most common infections treated worldwide each year and are primarily caused by uropathogenic E. coli (UPEC). Rising rates of antibiotic resistance among uropathogens have spurred consideration of alternative strategies such as bacteriophage (phage) therapy; however, phage-bacterial interactions within the urinary environment are poorly defined. Here, we assess the activity of two phages, HP3 and ES17, against clinical UPEC isolates using in vitro and in vivo models of UTI. In both bacteriologic medium and pooled human urine, we identified phage resistance arising within the first 6-8 hours of coincubation. Whole genome sequencing revealed that UPEC resistant to HP3 and ES17 harbored mutations in genes involved in lipopolysaccharide (LPS) biosynthesis. These mutations coincided with several in vitro phenotypes, including alterations to adherence to and invasion of human bladder epithelial HTB-9 cells, and increased biofilm formation. Interestingly, these phage-resistant UPEC demonstrated reduced growth in pooled human urine, which could be partially rescued by nutrient supplementation, and were more sensitive to several outer membrane targeting antibiotics than parental strains. Additionally, these phage-resistant UPEC were attenuated in a murine UTI model. In total, our findings suggest that while resistance to phages, such as LPS-targeted HP3 and ES17, may readily arise in the urinary environment, phage resistance is accompanied by fitness costs rendering UPEC more susceptible to host immunity or antibiotics. IMPORTANCEUTIs are one of the most common causes of outpatient antibiotic use, and rising antibiotic resistance threatens the ability to control these infections unless alternative treatments are developed. Bacteriophage (phage) therapy is gaining renewed interest, however, much like antibiotics, bacteria can readily become resistant to phage. For successful UTI treatment, we must predict how bacteria will evade killing by phage and identify the downstream consequences of phage-resistant bacterial infections. In our current study, we found that while phage-resistant mutant bacteria quickly emerged, these mutations left bacteria less capable of growing in human urine and colonizing the murine bladder. These results suggest that phage therapy poses a viable UTI treatment if phage resistance confers fitness costs for the uropathogen. These results have implications for developing cocktails of phage with multiple different bacterial targets, each of which is only evaded at the cost of bacterial fitness.

microbiology↗

bioRxiv2020

Targeting of Mammalian Glycans Enhances Phage Predation in the Gastrointestinal Tract

The human mucosal surface consists of a eukaryotic epithelium, a prokaryotic microbiota, and a carbohydrate-rich interface that separates them. Bacteriophage parasitize the prokaryotes but are not known to associate with eukaryotic cells. In the gastrointestinal tract, the interaction of these two domains influences the health of the host, especially colonization with invasive pathobionts. Antibiotics may be used but they also kill protective commensals and lack the physio-chemical properties to be specifically and optimally active in this complex milieu. Here, we report a novel phage whose lytic cycle is enhanced in intestinal environments. The enhanced activity is encoded in its tail fiber gene, whose protein product binds human heparan sulfated proteoglycans and localizes the phage to the epithelial cell surface, thereby positioning it near its bacterial host, a type of locational targeting mechanism. This finding offers the prospect of developing epithelial-targeting phage to selectively remove invasive pathobiont species from mucosal surfaces. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/212829v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@13219f6org.highwire.dtl.DTLVardef@469d7borg.highwire.dtl.DTLVardef@d31e0org.highwire.dtl.DTLVardef@458e14_HPS_FORMAT_FIGEXP M_FIG C_FIG Model showing (1) mucins from the intestinal mucus layer inhibit phage infection, (2) phage ES17 can bind to mucin and utilize other intestinal glycans as a receptor to infect and kill mucus-coated bacteria, and (3) phages like ES17 can be utilized to coat the intestinal epithelium by binding heparan sulfate glycans to protect from invasive pathogen infection.

microbiology↗

bioRxiv2020

Zika virus replicates in skeletal muscle contributing to peripheral viral amplification prior to reach neural tissue

Zika virus (ZIKV) infections are still a worldwide concern due to the severity of neurological outcomes. ZIKV neurotropism is well characterized, but peripheral tissue could be sites of viral amplification, contributing to endothelial-barrier crossing and access to peripheral nerves. During acute and late phases of infection, ZIKV can be detected in several body fluids, eyes, testis and vagina. However, the importance of initial replication sites for the establishment of infection and viral spread remain unknown. Here we demonstrated that ZIKV replicates primarily in human muscle precursor cells, resulting in cell death and inhibition of myogenesis. ZIKV also replicates in fetal muscle after maternal transmission and in infected neonate mice, inducing lesions and inflammation. Muscle was an important site of viral amplification, sustaining higher peripheral viral loads than liver and spleen. In addition, ZIKV showed rapid and sustained replication kinetics in muscle even before replication in the neural tissues, persisting until 16 days post infection. Our results highlight the importance of muscle in ZIKV pathogenesis as a peripheral site of viral amplification which may contribute to ZIKV reaching neural structures. Author SummaryZika Virus (ZIKV) neurotropism and its deleterious effects on central nervous system have been well characterized. But, investigations of the initial replication sites for the establishment of infection and viral spread to neural tissues remain under explored. Here we demonstrated that ZIKV replicates primarily in human skeletal muscle precursor cells, resulting in cell death and disrupted myogenesis. ZIKV also replicates in muscle of fetus and neonate mice inducing muscle damage and inflammation. Muscle replication occurs before amplification in peripheral nerves and brain, contributing to the increase of peripheral ZIKV load and dissemination. In addition, ZIKV RNA still been detected in skeletal muscle at late stages of infection. Overall, our findings showed that skeletal muscle is involved in ZIKV pathogenesis, contributing to a broader understanding of ZIKV infection. Thus, opens new aspects in the investigation of the long-term consequence of early infection.

microbiology↗

bioRxiv2020

Phage-Antibiotic Synergy Is Driven By A Unique Combination Of Antibacterial Mechanism Of Action And Stoichiometry

The continued rise in antibiotic resistance is precipitating a medical crisis. Bacteriophage (phage) has been hailed as one possible therapeutic option to augment the efficacy of antibiotics. However, only a handful of studies have addressed the synergistic relationship between phage and antibiotics. Here, we report a comprehensive analysis of phage-antibiotic interaction that evaluates synergism, additivism, and antagonism for all classes of antibiotics across clinically achievable stoichiometries. We combined an optically-based real-time microtiter plate readout with a matrix-like heatmap of treatment potencies to measure phage and antibiotic synergy (PAS), a process we term synography. Phage-antibiotic synography was performed against a pandemic drug-resistant clonal group of E. coli (ExPEC) with antibiotic levels blanketing the minimum inhibitor concentration (MIC) across seven orders of viral titers. Our results suggest that, under certain conditions, phages provide an adjuvating effect by lowering the MIC for drug-resistant strains. Furthermore, synergistic and antagonistic interactions are highly dependent on the mechanism of bacterial inhibition by the class of antibiotic paired to the phage, and when synergism is observed, it suppresses the emergence of resistant cells. Host conditions that simulate the infection environment, including serum and urine, suppress PAS in a bacterial growth-dependent manner. Lastly, phage burst size seems to be a significant driver of synergism. Collectively, this data suggests lytic phages can resuscitate an ineffective antibiotic for previously resistant bacteria, while also synergize with antibiotics in a class-dependent manner, processes that may be dampened by lower bacterial growth rates found in host environments. Significance StatementBacteriophage (phage) therapy is a promising approach to combat the rise of multi-drug resistant bacteria. Currently, the preferred clinical modality is to pair phage with an antibiotic, a practice thought to improve efficacy. However, antagonism between phage and antibiotics has been reported, the choice of phage and antibiotic is not often empirically determined, and the effect of the host factors on the effectiveness is unknown. Here, we interrogate phage-antibiotic interactions across antibiotics with different mechanisms of action. Our results suggest that phage can lower the working MIC for bacterial strains already resistant to the antibiotic, is dependent on the antibiotic class and stoichiometry of the pairing, and is dramatically influenced by the host microenvironment.

microbiology↗