Diene incorporation by a dehydratase domain variant in modular polyketide synthases.

Modular polyketide synthases (PKSs) are biosynthetic assembly lines that construct structurally diverse natural products with wide-ranging applications in medicine and agriculture. Various mechanisms contribute to structural diversification during PKS-mediated chain assembly, including dehydratase (DH) domain-mediated elimination of water from R and S-configured 3-hydroxy-thioesters to introduce E- and Z-configured carbon-carbon double bonds, respectively. Here we report the discovery of a DH domain variant that catalyzes the sequential elimination of two molecules of water from a (3R, 5S)-3,5-dihydroxy thioester during polyketide chain assembly, introducing a conjugated E,Z-diene into various modular PKS products. We show that the reaction proceeds via a (2E, 5S)-2-enoyl-5-hydroxy-thioester intermediate and involves an additional universally conserved histidine residue that is absent from the active site of most conventional DH domains. These findings expand the diverse range of chemistries mediated by DH-like domains in modular PKSs, highlighting the catalytic versatility of the double hotdog fold.

A Flavin-Dependent Decarboxylase-Dehydrogenase-Monooxygenase Assembles the Warhead of α,ß-Epoxyketone Proteasome Inhibitors.

The α,ß-epoxyketone proteasome inhibitor TMC-86A was discovered as a previously unreported metabolite of Streptomyces chromofuscus ATCC49982, and the gene cluster responsible for its biosynthesis was identified via genome sequencing. Incorporation experiments with [(13)C-methyl]l-methionine implicated an α-dimethyl-ß-keto acid intermediate in the biosynthesis of TMC-86A. Incubation of the chemically synthesized α-dimethyl-ß-keto acid with a purified recombinant flavin-dependent enzyme that is conserved in all known pathways for epoxyketone biosynthesis resulted in formation of the corresponding α-methyl-α,ß-epoxyketone. This transformation appears to proceed via an unprecedented decarboxylation-dehydrogenation-monooxygenation cascade. The biosynthesis of the TMC-86A warhead is completed by cytochrome P450-mediated hydroxylation of the α-methyl-α,ß-epoxyketone.

Dynamics of skeletal muscle-resident stem cells during myogenesis in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease in which extraskeletal (heterotopic) bone forms within tissues such as skeletal muscles, often in response to injury. Mutations in the BMP type I receptor ACVR1/ALK2 cause FOP by increasing BMP pathway signaling. In contrast to the growing understanding of the inappropriate formation of bone tissue within the muscle in FOP, much is still unknown about the regenerative capacity of adult diseased muscles. Utilizing an inducible ACVR1R206H knock-in mouse, we found that injured Acvr1R206H/+ skeletal muscle tissue regenerates poorly. We demonstrated that while two resident stem cell populations, muscle stem cells (MuSCs) and fibro/adipogenic progenitors (FAPs), have similar proliferation rates after injury, the differentiation potential of mutant MuSCs is compromised. Although MuSC-specific deletion of the ACVR1R206H mutation does not alter the regenerative potential of skeletal muscles in vivo, Acvr1R206H/+ MuSCs form underdeveloped fibers that fail to fuse in vitro. We further determined that FAPs from Acvr1R206H/+ mice repress the MuSC-mediated formation of Acvr1R206H/+ myotubes in vitro. These results identify a previously unrecognized role for ACVR1R206H in myogenesis in FOP, via improper interaction of tissue-resident stem cells during skeletal muscle regeneration.

Application of Data Science to Quantify the Effect of Propofol Infusion on Postoperative Nausea and Vomiting.

The effectiveness of propofol infusion on postoperative nausea and vomiting (PONV) is poorly understood in relation to various patient and procedure characteristics. This retrospective cohort study aimed to quantify the effectiveness of propofol infusion when administered either via total intravenous administration (TIVA) or combined intravenous anesthesia (CIVA) with inhalational agents on PONV. The relationship between propofol infusion and PONV was characterized controlling for patient demographics, procedure characteristics, PONV risk factors, and antiemetic drugs in adult patients (age ≥18 years) undergoing general anesthesia. Learned coefficients from multivariate regression models were reported as "lift" which represents the percentage change in the base likelihood of observing PONV if a variable is present versus absent. In a total of 41,490 patients, models showed that propofol infusion has a naive effect on PONV with a lift of -41% (P < .001) when using TIVA and -17% (P < .001) when using CIVA. Adding interaction terms to the model resulted in the loss of statistical significance in these relationships (lift of -30%, P = .23, when using TIVA, and -42%, P = .36, when using CIVA). It was further found that CIVA/TIVA are ineffective in short cases (CIVA * short surgery duration: lift = 49%, P < .001 and TIVA * short surgery duration: lift = 56%, P < .001).

A SARS-CoV-2 antigen rapid diagnostic test for resource limited settings.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 disease. RT-qPCR has been the primary method of diagnosis; however, the required infrastructure is lacking in many developing countries and the virus has remained a global challenge. More inexpensive and immediate test methods are required to facilitate local, regional, and national management strategies to re-open world economies. Here we have developed a SARS-CoV-2 antigen test in an inexpensive lateral flow format to generate a chromatographic result identifying the presence of the SARS-CoV-2 antigen, and thus an active infection, within a patient anterior nares swab sample. Our 15-min test requires no equipment or laboratory infrastructure to administer with a limit of detection of 2.0 × 102 TCID50/mL and 87.5% sensitivity, 100% specificity when tested against 40 known positive and 40 known negative patient samples established by a validated RT-qPCR test.

Nanoliter high-throughput PCR for DNA and RNA profiling.

The increasing emphasis in life science research on utilization of genetic and genomic information underlies the need for high-throughput technologies capable of analyzing the expression of multiple genes or the presence of informative single nucleotide polymorphisms (SNPs) in large-scale, population-based applications. Human disease research, disease diagnosis, personalized therapeutics, environmental monitoring, blood testing, and identification of genetic traits impacting agricultural practices, both in terms of food quality and production efficiency, are a few areas where such systems are in demand. This has stimulated the need for PCR technologies that preserves the intrinsic analytical benefits of PCR yet enables higher throughputs without increasing the time to answer, labor and reagent expenses and workflow complexity. An example of such a system based on a high-density array of nanoliter PCR assays is described here. Functionally equivalent to a microtiter plate, the nanoplate system makes possible up to 3,072 simultaneous end-point or real-time PCR measurements in a device, the size of a standard microscope slide. Methods for SNP genotyping with end-point TaqMan PCR assays and quantitative measurement of gene expression with SYBR Green I real-time PCR are outlined and illustrative data showing system performance is provided.

A dual transacylation mechanism for polyketide synthase chain release in enacyloxin antibiotic biosynthesis.

Polyketide synthases assemble diverse natural products with numerous important applications. The thioester intermediates in polyketide assembly are covalently tethered to acyl carrier protein domains of the synthase. Several mechanisms for polyketide chain release are known, contributing to natural product structural diversification. Here, we report a dual transacylation mechanism for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii. A non-elongating ketosynthase domain transfers the polyketide chain from the final acyl carrier protein domain of the synthase to a separate carrier protein, and a non-ribosomal peptide synthetase condensation domain condenses it with (1S,3R,4S)-3,4-dihydroxycyclohexane carboxylic acid. Molecular dissection of this process reveals that non-elongating ketosynthase domain-mediated transacylation circumvents the inability of the condensation domain to recognize the acyl carrier protein domain. Several 3,4-dihydroxycyclohexane carboxylic acid analogues can be employed for chain release, suggesting a promising strategy for producing enacyloxin analogues.

Social Avoidance Emerges in Infancy and Persists into Adulthood in Fragile X Syndrome.

Fragile X syndrome (FXS) is characterized by both social approach and social avoidance. However, the age of emergence and developmental trajectory of social avoidance has not been examined. This study investigates the longitudinal developmental trajectory and dynamic nature of social avoidance in males with FXS from infancy through young adulthood (n = 191). Multiple facets of social avoidance were collected using the Social Avoidance Scale (Roberts et al. 2007, 2009). Overall, 81% of males with FXS displayed social avoidance, which emerged during infancy, increased in severity across childhood, and stabilized through adolescence and early adulthood. An exaggerated "warm up" effect was also observed in FXS. This study delineates the complex profile of social avoidance, a common and impairing behavioral feature of FXS.

Infant Social Avoidance Predicts Autism but Not Anxiety in Fragile X Syndrome.

Objective: Autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), and anxiety are three of the most common childhood psychiatric disorders. Early trajectories of social avoidance have been linked with these psychiatric disorders in previous studies, but it remains unclear how social avoidance differentially predicts comorbid disorders in a high-risk genetic subgroup. Here, we delineate the association between trajectories of social avoidance from infancy and subsequent ASD, ADHD, and anxiety outcomes at preschool in children with fragile X syndrome (FXS), a well-characterized single-gene disorder highly associated with social avoidance as well as elevated rates of ASD, ADHD, and anxiety. Method: Males with FXS (n = 78) aged 4-62 months participated in a longitudinal study resulting in 201 assessments. The Social Avoidance Scale (SAS) documented socially avoidant behaviors from infancy in three domains-physical movement, facial expression, and eye contact during both the first minute and the last hour of an interaction. ASD, ADHD, and anxiety symptom outcomes at preschool were measured via parent-report questionnaires. Results: Increased social avoidance across infancy and preschool predicted elevated ASD symptom severity but reduced ADHD and anxiety symptom severity in males with FXS. Conclusion: ASD, ADHD, and anxiety symptoms relate inconsistently to social avoidance behaviors, providing new insight toward the debate of independence or overlap among these disorders in FXS and other disorders (i.e., ASD). The results suggest that the nuanced profile of the developmental and temporal aspects of social avoidance may inform more the accuracy of differential diagnoses of comorbid psychiatric disorders in FXS.

An unusual Burkholderia gladioli double chain-initiating nonribosomal peptide synthetase assembles 'fungal' icosalide antibiotics.

Burkholderia is a multi-talented genus of Gram-negative bacteria, which in recent years has become increasingly recognised as a promising source of bioactive natural products. Metabolite profiling of Burkholderia gladioli BCC0238 showed that it produces the asymmetric lipopeptidiolide antibiotic icosalide A1, originally isolated from a fungus. Comparative bioinformatics analysis of several genome-sequenced B. gladioli isolates identified a gene encoding a nonribosomal peptide synthase (NRPS) with an unusual architecture that was predicted to be responsible for icosalide biosynthesis. Inactivation of this gene in B. gladioli BCC0238 abolished icosalide production. PCR analysis and sequencing of total DNA from the original fungal icosalide A1 producer revealed it has a B. gladioli strain associated with it that harbours an NRPS with an identical architecture to that responsible for icosalide A1 assembly in B. gladioli BCC0238. Sequence analysis of the icosalide NRPS indicated that it contains two chain-initiating condensation (CI) domains. One of these is appended to the N-terminus of module 1 - a common architecture for NRPSs involved in lipopeptide assembly. The other is embedded in module 3, immediately downstream of a putative chain-elongating condensation domain. Analysis of the reactions catalysed by a tridomain construct from module 3 of the NRPS using intact protein mass spectrometry showed that the embedded CI domain initiates assembly of a second lipopeptide chain, providing key insights into the mechanism for asymmetric diolide assembly.

Nanoliter high throughput quantitative PCR.

Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.

The Yaf9 component of the SWR1 and NuA4 complexes is required for proper gene expression, histone H4 acetylation, and Htz1 replacement near telomeres.

Yaf9, Taf14, and Sas5 comprise the YEATS domain family in Saccharomyces cerevisiae, which in humans includes proteins involved in acute leukemias. The YEATS domain family is essential, as a yaf9Delta taf14Delta sas5Delta triple mutant is nonviable. We verify that Yaf9 is a stable component of NuA4, an essential histone H4 acetyltransferase complex. Yaf9 is also associated with the SWR1 complex, which deposits the histone H2A variant Htz1. However, the functional contribution of Yaf9 to these complexes has not been determined. Strains lacking YAF9 are sensitive to DNA-damaging agents, cold, and caffeine, and the YEATS domain is required for full Yaf9 function. NuA4 lacking Yaf9 retains histone acetyltransferase activity in vitro, and Yaf9 does not markedly reduce bulk H4 acetylation levels, suggesting a role for Yaf9 in the targeting or regulation of NuA4. Interestingly, yaf9Delta strains display reduced transcription of genes near certain telomeres, and their repression is correlated with reduced H4 acetylation, reduced occupancy by Htz1, and increased occupancy by the silencing protein Sir3. Additionally, the spectra of phenotypes, genes, and telomeres affected in yaf9Delta and htz1Delta strains are significantly similar, further supporting a role for Yaf9 in Htz1 deposition. Taken together, these data indicate that Yaf9 may function in NuA4 and SWR1 complexes to help antagonize silencing near telomeres.

The mitochondrial message-specific mRNA protectors Cbp1 and Pet309 are associated in a high-molecular weight complex.

In Saccharomyces cerevisiae, the nuclear-encoded protein Cbp1 promotes stability and translation of mitochondrial cytochrome b transcripts through interaction with the 5' untranslated region. Fusion of a biotin binding peptide tag to the C terminus of Cbp1 has now allowed detection in mitochondrial extracts by using peroxidase-coupled avidin. Cbp1 is associated with the mitochondrial membranes when high ionic strength extraction conditions are used. However, the protein is easily solubilized by omitting salt from the extraction buffer, which suggests Cbp1 is loosely associated with the membrane through weak hydrophobic interactions. Gel filtration analysis and blue native PAGE showed that Cbp1 is part of a single 900,000-Da complex. The complex was purified using the biotin tag and a sequence-specific protease cleavage site. In addition to Cbp1, the complex contains several polypeptides of molecular weights between 113 and 40 kDa. Among these, we identified another message-specific factor, Pet309, which promotes the stability and translation of mitochondrial cytochrome oxidase subunit I mRNA. A hypothesis is presented in which the Cbp1-Pet309 complex contains several message-specific RNA binding proteins and links transcription to translation of the mRNAs at the membrane.

Substrate selectivity of an isolated enoyl reductase catalytic domain from an iterative highly reducing fungal polyketide synthase reveals key components of programming.

A cis-acting enoyl reductase (ER) catalytic domain was isolated from a fungal highly reducing iterative polyketide synthase (HR-iPKS) for the first time and studied in vitro. The ER from the squalestatin tetraketide synthase forms a discrete dimeric protein in solution. The ER shows broad substrate selectivity, reducing enoyl species including both natural and unnatural substrates. Pantetheine-bound substrate thiolesters reacted much faster than the corresponding SNAC thiolesters. The unnatural substrates included Z-olefins, 2-ethyl olefins and pentaketides. Methylation of the substrate modifies the activity of the ER such that the 2,4-dimethyl oct-2-enoyl substrate fits into the active site but cannot be reduced. A new NMR-based assay was developed for the direct observation of the stereochemical preferences at the 4' position of the NADPH cofactor and the C-2 and C-3 positions of the substrates. The assay reveals that the fungal iPKS ER-catalysed reaction is stereochemically identical to that of the vertebrate FAS (vFAS) at the cofactor 4' position and the substrate 3-position, but the high stereoselectivity displayed by intact SQTKS is lost such that reprotonation at the 2-position is unselective by the isolated ER. A 3D model of ER was consistent with these observations and showed that the ER may sequester its final substrate to prevent further chain extension. The results support a developing model for programming by HR-iPKS in which competition for substrates between restrictive and permissive catalytic domains chaperones the growing polyketide to completion, while allowing for errors and evolution.

Molecular rules governing de novo methylation in cancer.

De novo methylation of CpG islands is seen in many cancers, but the general rules governing this process are not known. By analyzing DNA from tumors, as well as normal tissues, and by utilizing a range of published data, we have identified a universal set of tumor targets, each with its own "coefficient" of methylation that is largely correlated with its inherent relative ability to recruit polycomb. This pattern is initially formed by a slow process of de novo methylation that occurs during aging and then undergoes expansion early in tumorigenesis, where we hypothesize that it may act as an inhibitor of development-associated gene activation.

Aberrant DNA methylation in ES cells.

Both mouse and human embryonic stem cells can be differentiated in vitro to produce a variety of somatic cell types. Using a new developmental tracing approach, we show that these cells are subject to massive aberrant CpG island de novo methylation that is exacerbated by differentiation in vitro. Bioinformatics analysis indicates that there are two distinct forms of abnormal de novo methylation, global as opposed to targeted, and in each case the resulting pattern is determined by molecular rules correlated with local pre-existing histone modification profiles. Since much of the abnormal methylation generated in vitro appears to be stably maintained, this modification may inhibit normal differentiation and could predispose to cancer if cells are used for replacement therapy. Excess CpG island methylation is also observed in normal placenta, suggesting that this process may be governed by an inherent program.

Defective endochondral ossification-derived matrix and bone cells alter the lymphopoietic niche in collagen X mouse models.

Despite the appreciated interdependence of skeletal and hematopoietic development, the cell and matrix components of the hematopoietic niche remain to be fully defined. Utilizing mice with disrupted function of collagen X (ColX), a major hypertrophic cartilage matrix protein associated with endochondral ossification, our data identified a cytokine defect in trabecular bone cells at the chondro-osseous hematopoietic niche as a cause for aberrant B lymphopoiesis in these mice. Specifically, analysis of ColX transgenic and null mouse chondro-osseous regions via micro-computed tomography revealed an altered trabecular bone environment. Additionally, cocultures with hematopoietic and chondro-osseous cell types highlighted impaired hematopoietic support by ColX transgenic and null mouse derived trabecular bone cells. Further, cytokine arrays with conditioned media from the trabecular osteoblast cocultures suggested an aberrant hematopoietic cytokine milieu within the chondro-osseous niche of the ColX deficient mice. Accordingly, B lymphopoiesis was rescued in the ColX mouse derived trabecular osteoblast cocultures with interlukin-7, stem cell factor, and stromal derived factor-1 supplementation. Moreover, B cell development was restored in vivo after injections of interlukin-7. These data support our hypothesis that endrochondrally-derived trabecular bone cells and matrix constituents provide cytokine-rich niches for hematopoiesis. Furthermore, this study contributes to the emerging concept that niche defects may underlie certain immuno-osseous and hematopoietic disorders.

Altered matrix at the chondro-osseous junction leads to defects in lymphopoiesis.

The collagen X transgenic and null (ColX-Tg/KO) mice have revealed a link between endochondral ossification (EO) and hematopoiesis, and thus serve as model systems to study hematopoietic niches. The altered collagen X function in ColX-Tg/KO mice resulted not only in skeletal defects, which included changes in growth plate ultrastructure, altered localization of heparan sulfate proteoglycans (HSPG), and reduced trabecular bone, but also in hematopoietic defects, which included reduced B lymphocyte numbers throughout life without associated increases in B cell apoptosis. Consequently, the ColX-Tg/KO mice exhibited diminished in vitro and in vivo immune responses. Moreover, reduced expression of several hematopoietic and B lymphopoietic cytokines were measured from ColX-KO-derived hypertrophic chondrocyte and trabecular osteoblast cultures. Together, these data expand the current hematopoietic niche model by including the EO-derived extracellular matrix, for example, the collagen X/HSPG network, as well as the EO-derived hypertrophic chondrocytes and trabecular osteoblasts as hematopoietic signal mediating cells.

Congenic mice confirm that collagen X is required for proper hematopoietic development.

The link between endochondral skeletal development and hematopoiesis in the marrow was established in the collagen X transgenic (Tg) and null (KO) mice. Disrupted function of collagen X, a major hypertrophic cartilage matrix protein, resulted in skeletal and hematopoietic defects in endochondrally derived tissues. Manifestation of the disease phenotype was variable, ranging from perinatal lethality in a subset of mice, to altered lymphopoiesis and impaired immunity in the surviving mice. To exclude contribution of strain specific modifiers to this variable manifestation of the skeleto-hematopoietic phenotype, C57Bl/6 and DBA/2J collagen X congenic lines were established. Comparable disease manifestations confirmed that the skeleto-hematopoietic alterations are an inherent outcome of disrupted collagen X function. Further, colony forming cell assays, complete blood count analysis, serum antibody ELISA, and organ outgrowth studies established altered lymphopoiesis in all collagen X Tg and KO mice and implicated opportunistic infection as a contributor to the severe disease phenotype. These data support a model where endochondral ossification-specific collagen X contributes to the establishment of a hematopoietic niche at the chondro-osseous junction.