Biology subjects

Katz, A.

Publications and source records attributed to Katz, A..

18 recordsLinked to original sources

bioRxiv2026

Impaired lipoprotein secretion by APOE4 leads to lysosomal and mitochondrial dysfunction in human microglia

While Apolipoprotein E4 (APOE4) is the greatest known genetic risk factor for late-onset Alzheimers disease, its mechanistic role in the brain-resident macrophage, microglia, remains elusive. Microglia are important in the clearance of pathology in disease, heavily relying on lysosome functionality; therefore, we sought to understand the impact of APOE4 on microglial function. APOE44 microglia have been shown to have lipid accumulation, yet the mechanisms leading to this accumulation are unknown. Using induced pluripotent stem cell-derived microglia, we found that the APOE4 haplotype resulted in transcriptional state shifts in microglia, suppressing activated-response microglia (ARMs) and promoting a G2 senescent-like state. We found that APOE44 microglia accumulate cholesterol esters and provide less lipid support to fibroblast-induced neurons, decreasing their synaptic connections. APOE44 microglia secrete significantly less lipoproteins, leading to the accumulation of lipoproteins within the cells including the lysosomes. APOE44 microglia exhibit impaired lysosomal acidification and degradation capacity. Further, our results elucidated that APOE44 microglia are proinflammatory and shift away from fatty acid oxidation towards glycolysis, due to dysfunctional mitochondria. Taken together, our findings indicate that a loss-of-function in lipoprotein secretion drives intracellular lipid accumulation, including within lysosomes, ultimately disrupting the lysosome-endoplasmic reticulum-mitochondrial axis. This drives a proinflammatory and metabolically compromised microglial phenotype with impaired neuro-supportive functions. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/724612v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@18d6a2org.highwire.dtl.DTLVardef@b3644dorg.highwire.dtl.DTLVardef@17e3716org.highwire.dtl.DTLVardef@1529caf_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2026

pertTF: context-aware AI modeling for genome-scale and cross-system perturbation prediction

Predicting genetic perturbation responses at a single-cell level is central to building models for cell state and disease. However, existing approaches are limited on predicting phenotypic outcomes beyond expression changes and generalizing predictions across genome-scale perturbations in biologically relevant contexts. Here we introduce pertTF, a transformer-based single-cell genetic perturbation model. pertTF was trained from a unique dataset capturing single cell expressions profiles of 30 full gene knockouts across 14 relevant cell types during human pancreatic development and beta-cell differentiation. pertTF outperforms current methods in predicting expression changes of perturbing unseen genes in unseen cellular contexts. In addition, pertTF infers perturbation-induced shifts in cell identity and population composition, an important phenotypic outcome of perturbation in many physiology and disease settings. Through transfer learning, pertTF operates in physiologically relevant systems, including primary human islets, where large-scale perturbation experiments are challenging. The generalizability of pertTF is further demonstrated by in silico pooled and single-cell CRISPR screens, capturing critical regulators of stem cells and early pancreatic cell development. These results establish pertTF as a framework for integrating large-scale single-cell perturbation data with AI models to predict genetic perturbation effects across cellular systems and disease contexts.

bioinformatics↗

bioRxiv2025

Active Conformations of Neuronal Na+,K+-ATPase isoforms and a Disease-Causing Mutant

Na+,K+-ATPases establish and maintain the vital electrochemical gradients for Na+ and K+ across animal cell membranes. The protein is a ternary complex composed of , {beta} and FXYD subunits, of which isoforms that fine-tune transport properties are expressed in a tissue-specific fashion. Here we report cryo-EM structures under active ATPase turn-over conditions of the ubiquitously expressed human 1{beta}1FXYD1 and neuron-specific 3{beta}1FXYD1 isoform complexes and probe their specific functional and biophysical properties. The data provides an extensive insight into Na+-transport of ATP-activated enzyme through four distinct conformational states, including a long-sought sodium-bound phosphoenzyme intermediate, denoted [Na3]E2P. This hitherto elusive conformation reveals a crucial structural change that precedes Na+ release in the inward to outward (E1P-E2P) transition, within the general context of the sequential, active transport mechanism. We investigated and discuss the mechanism of the physiologically important differentiation in Na+ affinity of 3 compared to 1, the co-operative Na+ binding at the ion-binding sites, and the mechanistic aspects of cytoplasmic ion gating and extracellular Na+ release. Finally we present the first structures of a disease-causing mutant form of 3, associated with Alternating Hemiplegia of Childhood (Q140L). The mutation compromises a specific phospholipid-binding pocket and impedes polyunsaturated phospholipid-mediated stimulation of Na+,K+-ATPase activity.

biochemistry↗

bioRxiv2025

Prenatal Zika Virus Exposure Disrupts Social-Emotional Development and Cortical Visual Function in Infant Macaques

Prenatal exposure to Zika virus (ZIKV) results in a spectrum of outcomes, ranging from severe birth defects and early childhood developmental delays to no apparent deficits. However, the mechanisms underlying these outcomes, particularly in the context of different maternal infection conditions, remain poorly defined. In this study, we employed a translational rhesus macaque model of prenatal ZIKV infection to evaluate longitudinal visual and auditory development from 1 to 12 months of age and characterize neurodevelopmental outcomes at 12 months. Pregnant macaques, either flavivirus-naive or with prior dengue virus (DENV) exposure, were inoculated in the first trimester ([~]30 or 45 gestational days) with an Asian or African lineage of ZIKV or received a saline injection as controls. Maternal plasma viremia duration, viral RNA burden at the maternal-fetal interface, and neutralizing antibody titers did not differ between inoculation cohorts or controls. At 12 months, ZIKV-exposed infants demonstrated altered maternal attachment behaviors and reduced inhibition when approaching sensory stimuli compared to controls. Visual pathway function, assessed by electrophysiology, was significantly impaired at 3 months but normalized by 12 months. Hearing loss was more common among ZIKV-exposed infants, although not statistically significant. Developmental outcomes were associated with prenatal ZIKV exposure itself, independent of viremia duration, neutralizing antibody titer, viral lineage, or maternal DENV immunity, and were not mediated by visual impairment or hearing loss. These findings highlight that any prenatal ZIKV exposure can disrupt early neurobehavioral development and visual function, underscoring the need for prevention strategies focused on maternal infection and early intervention.

microbiology↗

bioRxiv2024

Elucidating early intestinal stem cell response to bacterial infection

Intestinal stem cells (ISCs) are the regenerative force of the gut epithelium. Lgr5+-ISC have been shown to respond to changes in their microenvironment by coping with different metabolites, adapting to caloric changes, and recovering from injury and inflammation. However, how pathogenic bacteria affect adult stem cell regeneration and, as a consequence, the overall tissue adaption to infection has yet to be explored in depth. Here, we interrogated early Lgr5+ ISC responses to an enteric intracellular pathogen by profiling individual IECs from the mouse small intestine. Utilizing GFP-labeled Salmonella enterica, we isolated intracellular invaded cells to elucidate invasion programs of epithelial cell subsets. In particular, we identified a Salmonella-specific infection signature comprised of antimicrobial peptide (AMP) genes, including the Defensin gene family. Our findings demonstrate that Salmonella enterica targets differentiated Paneth, enterocytes, and stem/progenitor cells at these early stages of infection. In response, a rapid Lg5+ ISC-driven cellular remodeling to enterocyte and Paneth lineages expressing AMP genes is initiated to combat the intruders. Importantly, we uncovered an ISC differentiation program via inflammasome activation to protect the crypt environment, while eliminating infected stem cells from the overall stem cell pool. This novel Lgr5+ stem cell defense mechanism not only protects the gut epithelium from persistent bacterial infection but also promotes tissue regeneration. We propose epithelial remodeling to AMP-secreting cells as a novel innate immune response to handle different gut stresses mediated by Lgr5+ ISCs to maintain organizational principles of gut homeostasis and physiology.

immunology↗

bioRxiv2023

Multi-target genome editing reduces polyphenol oxidase activity in wheat (Triticum aestivum L.) grains

Polyphenol oxidases (PPO) are dual activity metalloenzymes that catalyse the production of quinones. In plants, PPO activity may contribute to biotic stress resistance and secondary metabolism but is undesirable for food producers because it causes the discolouration and changes in flavour profiles of products during post-harvest processing. In wheat (Triticum aestivum L.), PPO released from the aleurone layer of the grain during milling results in the discolouration of flour, dough, and end-use products, reducing their value. Loss-of-function mutations in the PPO1 and PPO2 paralogous genes on homoeologous group 2 chromosomes confer reduced PPO activity in the wheat grain but limited natural variation and small intergenic distances between these genes complicates the selection of extremely low-PPO wheat varieties by recombination. In the current study, a CRISPR/Cas9 construct with one single guide RNA (sgRNA) targeting a conserved copper binding domain was used to edit all seven PPO1 and PPO2 genes in the spring wheat cultivar Fielder. Five of the seven edited T1 lines exhibited significant reductions in PPO activity, and T2 lines had PPO activity up to 86.7% lower than wild-type controls. In the elite winter wheat cultivars Guardian and Steamboat, which have five PPO1 and PPO2 genes, PPO activity was reduced by >90% in both T1 and T2 lines. This study demonstrates that multi-target editing at late stages of variety development could complement selection for beneficial alleles in crop breeding programmes by inducing novel variation in loci inaccessible to recombination.

plant biology↗

bioRxiv2023

ATP1A1-linked diseases require a malfunctioning protein product from one allele

Heterozygous germline variants in ATP1A1, the gene encoding the 1 subunit of the Na+/K+-ATPase (NKA), have been linked to diseases including primary hyperaldosteronism and the peripheral neuropathy Charcot-Marie-Tooth disease (CMT). ATP1A1 variants that cause CMT induce loss-of-function of NKA. This heterodimeric ({beta}) enzyme hydrolyzes ATP to establish transmembrane electrochemical gradients of Na+ and K+ that are essential for electrical signaling and cell survival. Of the 4 catalytic subunit isoforms, 1 is ubiquitously expressed and is the predominant paralog in peripheral axons. Human population sequencing datasets indicate strong negative selection against both missense and protein-null ATP1A1 variants. To test whether haploinsufficiency generated by heterozygous protein-null alleles are sufficient to cause disease, we tested the neuromuscular characteristics of heterozygous Atp1a1+/- knockout mice and their wildtype littermates, while also evaluating if exercise increased CMT penetrance. We found that Atp1a1+/- mice were phenotypically normal up to 18 months of age. Consistent with the observations in mice, we report clinical phenotyping of a healthy adult human who lacks any clinical features of known ATP1A1-related diseases despite carrying a protein-null early truncation variant, p.Y148*. Taken together, these results suggest that a malfunctioning gene product is required for disease induction by ATP1A1 variants and that if any pathology is associated with protein-null variants, they may display low penetrance or high age of onset.

physiology↗

bioRxiv2023

Interpersonal early adversity demonstrates dissimilarity from early socioeconomic disadvantage in the course of human brain development: A meta-analysis

It has been established that early-life adversity impacts brain development, but the role of development itself has largely been ignored. We take a developmentally-sensitive approach to examine the neurodevelopmental sequelae of early adversity in a preregistered meta-analysis of 27,234 youth (birth to 18-years-old), providing the largest group of adversity-exposed youth to date. Findings demonstrate that early-life adversity does not have an ontogenetically uniform impact on brain volumes, but instead exhibits age-, experience-, and region-specific associations. Relative to non-exposed comparisons, interpersonal early adversity (e.g., family-based maltreatment) was associated with initially larger volumes in frontolimbic regions until ~10-years-old, after which these exposures were linked to increasingly smaller volumes. By contrast, socioeconomic disadvantage (e.g., poverty) was associated with smaller volumes in temporal-limbic regions in childhood, which were attenuated at older ages. These findings advance ongoing debates regarding why, when, and how early-life adversity shapes later neural outcomes.

neuroscience↗

bioRxiv2021

CytoPro: A computational platform for accurate and robust assessment of cell contributions in bulk expression from diverse tissues and conditions

Cells are the quanta unit of biology and their relative composition in a tissue is the prime driver of bulk tissue gene expression variation. When there is no cell information, deconvolution is an effective tool to achieve cell resolution, which provides important information for learning disease complexity and its interactions with treatments, drugs and/or the environment in a wide variety of contexts. Here we present CytoPro, a production-level tissue and condition-specific deconvolution platform, based on a large collection of human tissue-specific signatures derived from single and sorted cells. CytoPro infer per-sample multiple cell-type composition, given input bulk gene expression. CytoPro includes a rigorous QC pipeline for learning, generating and selecting signatures and performs internal automated validation using multiple QC test criteria including: Comparison to ground truth cytometry and pure sorted cells data, performance evaluation using simulated data including robustness to noise as well as agreement with biological expectations in validation datasets regarding genes and cells. We demonstrate that CytoPro outperforms existing deconvolution tools, in both accuracy and robustness. By exploring multiple datasets with predefined disease phenotypes, and analyzing a use-case of biological treatment response, we show the ability of CytoPro to flush out relevant cell biology in real pathological conditions.

bioinformatics↗

bioRxiv2021

Enhanced translation of leaderless mRNAs under oxidative stress in Escherichia coli

The bacterial response to oxidative stress requires the adaptation of the proteome to the hostile environment. It has been reported that oxidative stress induces a strong and global inhibition of both, transcription and translation. Nevertheless, whereas it is well known that transcription of a small group of genes is induced thanks to transcription factors such as OxyR and SoxR, an equivalent mechanism has not been described for translation. Here we report that whereas canonical translation that depends on Shine Dalgarno recognition is inhibited by oxidative stress in Escherichia coli, the translation of leaderless mRNA (lmRNA) is enhanced under such conditions. Both, inhibition of canonical translation and enhancement of lmRNA translation, depend on the production of (p)ppGpp. We propose that such a mechanism would allow bacteria to rapidly adapt their proteome to hostile conditions and is, perhaps, a general strategy to confront strong stressful conditions. Significance statementThe regulation of translation (the production of proteins based on genetic information) is central for the adaptation to environmental changes. In Escherichia coli translation may begin through two alternative pathways. 1.- A canonical initiation that is well understood and is regulated mostly by changes in the accessibility of ribosomes to specific sequences and 2.- Initiation of leaderless mRNAs (lmRNAs) that lack these sequences and for which we do not understand the regulation process. Our results indicate that under oxidative stress, the production of (p)ppGpp in E. coli inhibits canonical translation and simultaneously enhances translation of lmRNAs, showing for the first time a natural condition where lmRNA translation is regulated and a role for (p)ppGpp in this process.

microbiology↗

bioRxiv2021

A personalized network framework reveals predictive axis of anti-TNF response across diseases

Personalized treatment of complex diseases has been mostly predicated on biomarker identification of one drug-disease combination at a time. Here, we used a novel computational approach termed Disruption Networks to generate a new data type, contextualized by cell-centered individual-level networks, that captures biology otherwise overlooked when performing standard statistics. The new data-type extends beyond the feature level space, to the relations space, by quantifying individual-level breaking or rewiring of cross-feature relations. Applying disruption network to dissect high-dimensional blood data, we discover and validate that the RAC1-PAK1 axis is predictive of anti-TNF response in inflammatory bowel disease. Intermediate monocytes, which correlate with the inflammatory state, play a key role in the RAC1-PAK1 responses, supporting their modulation as a therapeutic target. This axis also predicts response in rheumatoid arthritis, validated in three public cohorts. Our findings support blood-based drug response diagnostics across immune-mediated diseases, implicating common mechanisms of non-response.

systems biology↗

bioRxiv2021

Development of the Wheat Practical Haplotype Graph Database as a Resource for Genotyping Data Storage and Genotype Imputation

To improve the efficiency of high-density genotype data storage and imputation in bread wheat (Triticum aestivum L.), we applied the Practical Haplotype Graph (PHG) tool. The wheat PHG database was built using whole-exome capture sequencing data from a diverse set of 65 wheat accessions. Population haplotypes were inferred for the reference genome intervals defined by the boundaries of the high-quality gene models. Missing genotypes in the inference panels, composed of wheat cultivars or recombinant inbred lines genotyped by exome capture, genotyping-by-sequencing (GBS), or whole-genome skim-seq sequencing approaches, were imputed using the wheat PHG database. Though imputation accuracy varied depending on the method of sequencing and coverage depth, we found 93% imputation accuracy with 0.01x sequence coverage, which was only slightly lower than the accuracy obtained using the 0.5x sequence coverage (96.9%). Compared to Beagle, on average, PHG imputation was ~4% (p-value = 0.00027) more accurate, and showed 27% higher accuracy at imputing a rare haplotype introgressed from a wild relative into wheat. The reduced accuracy of imputation with GBS data (90.4%) is likely associated with the small overlap between GBS markers and the exome capture dataset, which was used for constructing PHG. The highest imputation accuracy was obtained with exome capture for the wheat D genome, which also showed the highest levels of linkage disequlibrium and proportion of identity-by-descent regions among accessions in our reference panel. We demonstrate that genetic mapping based on genotypes imputed using PHG identifies SNPs with a broader range of effect sizes that together explain a higher proportion of genetic variance for heading date and meiotic crossover rate compared to previous studies.

genomics↗

bioRxiv2020

GMO LABELING IN FOOD PRODUCTS IN MONTEVIDEO, URUGUAY

Montevideo establishes the mandatory labeling of foods containing genetically modified material through the Departmental Decree No. 36.554, positioning Uruguay within the 65 countries that have incorporated this type of regulation. The Food Regulation Service, in its role of sanitary police, and through its Laboratory of Bromatology, in agreement with the Food Molecular Traceability Laboratory (Faculty of Sciences, University of the Republic), carried out the analysis of 206 products made with ingredients derived from corn and/or soybean, during the 2015-2017 period, within the framework of compliance with the aforementioned Decree. The strategy used consisted of the application of molecular techniques (Real Time PCR), for the detection of common sequences present in the transgenic events of soybean and corn, and the subsequent quantification of the content of GM material, in relation to an established labeling threshold of 1%. As a result of this study, it was found that 36.9% of the analyzed foods presented sequences derived from genetically modified plant organisms (GMOs); and in 95% of the cases, its content exceeded the threshold established for its labeling. This study, constitutes the first approach to the knowledge of different transgenic elements distribution in food commercialized in Montevideo. These results provide valuable information to both the consumer, for decision making about the food to be consumed, and also for the official control organizations, which must enforce the regulations. This type of work has demonstrated, once again, the importance of the interrelation between academy and state agencies, in the generation of knowledge and in the implementation of new analytical methodologies, as well as in the training of qualified human resources and in the compliance with current regulations.

molecular biology↗

bioRxiv2020

Genomic analysis of the diversity, antimicrobial resistance and virulence potential of Campylobacter jejuni and Campylobacter coli strains from a private health care center in Central Chile

Campylobacter jejuni and Campylobacter coli are the leading cause of human gastroenteritis in the industrialized world and an emerging threat in developing countries. The incidence of campylobacteriosis in South America is greatly underestimated, mostly due to the lack of adequate diagnostic methods. Accordingly, there is limited genomic and epidemiological data from this region. In the present study, we performed a genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance of the largest collection of clinical C. jejuni and C. coli strains from Chile available to date (n=81), collected in 2017-2019 in Santiago, Chile. This culture collection accounts for over a third of the genome sequences available of clinical strains from South America. cgMLST analysis identified high genetic diversity as well as 13 novel STs and alleles in both C. jejuni and C. coli. Pangenome and virulome analyses showed a differential distribution of virulence factors, including both plasmid and chromosomally encoded T6SSs and T4SSs. Resistome analysis predicted widespread resistance to fluoroquinolones, but low rates of erythromycin resistance. This study provides valuable genomic and epidemiological data and highlights the need for further genomic epidemiology studies in Chile and other South American countries to better understand molecular epidemiology and antimicrobial resistance of this emerging intestinal pathogen. AUTHOR SUMMARYCampylobacter is the leading cause of bacterial gastroenteritis worldwide and an emerging and neglected pathogen in South America. In this study, we performed an in-depth analysis of the genome sequences of 69 C. jejuni and 12 C. coli clinical strains isolated from Chile, which account for over a third of the sequences from clinical strains available from South America. We identified a high genetic diversity among C. jejuni strains and the unexpected identification of clade 3 C. coli strains, which are infrequently isolated from humans in other regions of the world. Most strains harbored the virulence factors described for Campylobacter. While [~]40% of strains harbored mutation in the gyrA gene described to confer fluoroquinolone resistance, very few strains encoded the determinants linked to macrolide resistance, currently used for the treatment of campylobacteriosis. Our study contributes to our knowledge of this important foodborne pathogen providing valuable data from South America.

microbiology↗

bioRxiv2020

Non-CG methylation is superior to CG methylation in genome regulation

DNA methylation in plants occurs in CG, CHG, and CHH sites. While depletion of CG methylation in transposons is associated with ample transcriptional activation, it was mainly studied in species with limited non-CG methylation that is linked to CG methylation. Here we profiled transcription in the moss plant, Physcomitrella patens, that has robust non-CG methylation with similar symmetrical CG and CHG methylation levels. Separated contextual methylation mechanisms in Physcomitrella patens enabled generation of numerous context-specific hypomethylated mutants. Our transcriptome data show that specific elimination of CG methylation is fully complemented by non-CG methylation. Conversely, exclusive removal of non-CG methylation massively dysregulated genes and transposons. Moreover, CHG methylation silenced transposons stronger than CG methylation. Lastly, we found non-CG methylation as crucial for silencing CG-depleted transposons. These results demonstrate the potency of non-CG methylation in genome regulation and suggest that it evolved due to moderate silencing and/or rapid mutability of methylated CGs.

genomics↗

bioRxiv2019

Pharyngeal motor cortex grey matter abnormalities and retinal photoreceptor layer dysfunction in macaques exposed to Zika virus in utero

Congenital Zika virus (ZIKV) exposure results in a spectrum of disease ranging from severe birth defects to delayed onset neurodevelopmental deficits. ZIKV-related neuropathogenesis, predictors of birth defects, and neurodevelopmental deficits are not well defined in people. Here we assess the methodological and statistical feasibility of a congenital ZIKV exposure macaque model for identifying infant neurobehavior and brain abnormalities that may underlie neurodevelopmental deficits. We inoculated five pregnant macaques with ZIKV and mock-inoculated one macaque in the first trimester. Following birth, growth, ocular structure/function, brain structure, hearing, histopathology, and neurobehavior were quantitatively assessed during the first week of life. We identified the typical pregnancy outcomes of congenital ZIKV infection, with fetal demise and placental abnormalities. We estimated sample sizes needed to define differences between groups and demonstrated that future studies quantifying brain region volumes, retinal structure, hearing, and visual pathway function require a sample size of 14 animals per group (14 ZIKV, 14 control) to detect statistically significant differences in at least half of the infant exam parameters. Establishing the parameters for future studies of neurodevelopmental outcomes following congenital ZIKV exposure in macaques is essential for robust and rigorous experimental design.

microbiology↗

bioRxiv2019

Structural characterization of the PCV2d genotype at 3.3 A resolution reveals differences to PCV2a and PCV2b genotypes, a tetranucleotide, and an N-terminus near the icosahedral 3-fold axes

Porcine circovirus 2 (PCV2) is a T=1 non-enveloped icosahedral virus that has a major impact on the swine industry as an agent of porcine circovirus associate disease. PCV2 capsid protein sequences have been employed by others to provide a temporal description of the emerging genotypes. PCV2a is believed to be the earliest genotype and responsible for giving rise to PCV2b, which gives rise to PCV2d. The underlying mechanism responsible for the emerging genotypes is not understood. To determine if a change in the PCV2d capsid accompanies the emergence of this genotype, we determined the cryo-electron microscopy image reconstruction of PCV2d VLP at 3.3 [A] resolution and compared it to the previously reported PCV2a and PCV2b structures. Differences between the CD and GH loops identify structural changes that accompany the emergence of PCV2b from PCV2a, and PCV2d from PCV2b. We also model additional amino acids for the N-terminus near the icosahedral 3-fold axes of symmetry and a tetranucleotide between the 5- and 2-fold axes of symmetry. To interpret the sequence diversity that defines the PCV2 genotypes on a structural platform we have performed structure-based sequence comparison. Our analysis demonstrates that each genotype possesses a unique set of amino acids located on the surface of the capsid that experience a high degree of substitution. These substitutions may be a response to the PCV2 vaccination program. The structural difference between PCV2a, b and d genotypes indicate that it is important to determine the PCV2 capsid structure as the virus evolves into different genotypes.\n\nImportancePCV2 is a significant epidemic agricultural pathogen that is the causative agent of a variety of swine illnesses. PCV2 infections have significant economic impact in the swine industry and must be controlled by vaccination. Outbreaks in farms vaccinated for PCV2 suggest that improvements to the current vaccination programs are needed. Better understanding of the assembly, structure, replication and evolution of these viruses is necessary for production of improved vaccines. The ability of PCV2 to rapidly shift genotypes suggests that expression systems capable of rapidly producing large quantities of virus-like particles should be pursued. To these ends we have established a mammalian cell-based virus-like particle expression system and performed high resolution structural studies of a new PCV2 genotype. Differences between the structure of this genotype and earlier genotypes demonstrate that it is important to study the PCV2 structure as it shifts genotypes.

biophysics↗

bioRxiv2017

Authentic Enzyme Intermediates Captured "on-the-fly" by Mix-and-Inject Serial Crystallography

Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Here, we demonstrate a general method for capturing enzyme catalysis in-action by mix-and-inject serial crystallography. Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis -lactamase with the 3rd generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation on the millisecond to second time scales including the crossover from transition state kinetics to steady-state kinetics.\n\nSynopsisAn enzymatically catalyzed reaction is initiated by diffusion based mixing of substrate and followed at runtime by time-resolved serial crystallography using a free electron laser.

biophysics↗