Assessment of environmental safety and protective efficacy of O-antigen deficient DIVA capable Salmonella Enteritidis against chicken salmonellosis.

In this study, we incorporated deletion of the O-antigen ligase gene to an attenuated Salmonella Enteritidis (SE) strain, JOL919 (SE PS; Δlon ΔcpxR), using the Lambda-Red recombination method and evaluated the safety and immunological aspects of the novel genotype, JOL2381 (SE VS: Δlon, ΔcpxR, ΔrfaL). Assessment of fecal shedding and organ persistence following administration via oral and IM routes revealed that the SE VS was safer than its parent strain, SE PS. Immunological assays confirmed that immunization via the oral route with SE PS was superior to the SE VS. However, chickens immunized with SE PS and SE VS strains via the IM route showed higher humoral and cell-mediated immune responses. Compared to PBS control, the IM route of immunization with SE VS resulted in a higher IgY antibody titer and expansion of CD4+ and CD8+ T-cell populations, which resulted in the clearance of Salmonella from the liver and splenic tissues. Furthermore, deletion of the O-antigen ligase gene caused lower production of LPS-specific antibodies in the host, promoting DIVA functionality and making it a plausible candidate for field utilization. Due to significant protection, high attenuation, and environmental safety concerns, the present SE VS strain is an ideal choice to prevent chicken salmonellosis and ensure public health.

Assessment of the Cardiac Noncoding Transcriptome by Single-Cell RNA Sequencing Identifies FIXER, a Conserved Profibrogenic Long Noncoding RNA.

BACKGROUND: Cardiac fibroblasts have crucial roles in the heart. In particular, fibroblasts differentiate into myofibroblasts in the damaged myocardium, contributing to scar formation and interstitial fibrosis. Fibrosis is associated with heart dysfunction and failure. Myofibroblasts therefore represent attractive therapeutic targets. However, the lack of myofibroblast-specific markers has precluded the development of targeted therapies. In this context, most of the noncoding genome is transcribed into long noncoding RNAs (lncRNAs). A number of lncRNAs have pivotal functions in the cardiovascular system. lncRNAs are globally more cell-specific than protein-coding genes, supporting their importance as key determinants of cell identity. METHODS: In this study, we evaluated the value of the lncRNA transcriptome in very deep single-cell RNA sequencing. We profiled the lncRNA transcriptome in cardiac nonmyocyte cells after infarction and probed heterogeneity in the fibroblast and myofibroblast populations. In addition, we searched for subpopulation-specific markers that can constitute novel targets in therapy for heart disease. RESULTS: We demonstrated that cardiac cell identity can be defined by the sole expression of lncRNAs in single-cell experiments. In this analysis, we identified lncRNAs enriched in relevant myofibroblast subpopulations. Selecting 1 candidate we named FIXER (fibrogenic LOX-locus enhancer RNA), we showed that its silencing limits fibrosis and improves heart function after infarction. Mechanitically, FIXER interacts with CBX4, an E3 SUMO protein ligase and transcription factor, guiding CBX4 to the promoter of the transcription factor RUNX1 to control its expression and, consequently, the expression of a fibrogenic gene program.. FIXER is conserved in humans, supporting its translational value. CONCLUSIONS: Our results demonstrated that lncRNA expression is sufficient to identify the various cell types composing the mammalian heart. Focusing on cardiac fibroblasts and their derivatives, we identified lncRNAs uniquely expressed in myofibroblasts. In particular, the lncRNA FIXER represents a novel therapeutic target for cardiac fibrosis.

Longitudinal assessment of reactivity and affinity profile of anti-Jo1 autoantibodies to distinct HisRS domains and a splice variant in a cohort of patients with myositis and anti-synthetase syndrome.

BACKGROUND: To address the reactivity and affinity against histidyl-transfer RNA synthetase (HisRS) autoantigen of anti-Jo1 autoantibodies from serum and bronchoalveolar lavage fluid (BALF) in patients with idiopathic inflammatory myopathies/anti-synthetase syndrome (IIM/ASSD). To investigate the associations between the reactivity profile and clinical data over time. METHODS: Samples and clinical data were obtained from (i) 25 anti-Jo1+ patients (19 sera with 16 longitudinal samples and 6 BALF/matching sera at diagnosis), (ii) 29 anti-Jo1- patients (25 sera and 4 BALF/matching sera at diagnosis), and (iii) 27 age/gender-matched healthy controls (24 sera and 3 BALF/matching sera). Reactivity towards HisRS full-length (HisRS-FL), three HisRS domains (WHEP, antigen binding domain (ABD), and catalytic domain (CD)), and the HisRS splice variant (SV) was tested. Anti-Jo1 IgG reactivity was evaluated by ELISA and western blot using IgG purified from serum by affinity chromatography. In paired serum-BALF, anti-Jo1 IgG and IgA reactivity was analyzed by ELISA. Autoantibody affinity was measured by surface plasmon resonance using IgG purified from sera. Correlations between autoantibody reactivity and clinical data were evaluated at diagnosis and longitudinally. RESULTS: Anti-Jo1 IgG from serum and BALF bound HisRS-FL, WHEP, and SV with high reactivity at the time of diagnosis and recognized both conformation-dependent and conformation-independent HisRS epitopes. Anti-HisRS-FL IgG displayed high affinity early in the disease. At the time of IIM/ASSD diagnosis, the highest autoantibody levels against HisRS-FL were found in patients ever developing interstitial lung disease (ILD) and arthritis, but with less skin involvement. Moreover, the reactivity of anti-WHEP IgG in BALF correlated with poor pulmonary function. Levels of autoantibodies against HisRS-FL, HisRS domains, and HisRS splice variant generally decreased over time. With some exceptions, longitudinal anti-HisRS-FL antibody levels changed in line with ILD activity. CONCLUSION: High levels and high-affinity anti-Jo1 autoantibodies towards HisRS-FL were found early in disease in sera and BALF. In combination with the correlation of anti-HisRS-FL antibody levels with ILD and ILD activity in longitudinal samples as well as of anti-WHEP IgG in BALF with poor pulmonary function, this supports the previously raised hypothesis that the lung might have a role in the immune reaction in anti-Jo1-positive patients.

Ganga river water quality assessment using combined approaches: physico-chemical parameters and cyanobacterial toxicity detection with special reference to microcystins and molecular characterization of microcystin synthetase (mcy) genes carrying cyanobacteria.

Water quality assessment relies mostly on physico-chemical-based characterization; however, eutrophication and climate change advocate the abundance of toxic microcystins (MCs) producing cyanobacteria as emerging bio-indicator. In the present study, a spatial-temporal analysis was carried out at ten sampling sites of Prayagraj and Varanasi during June 2017 and March 2018 to determine the Ganga River water quality using physico-chemical parameters, cyanobacteria diversity, detection of MCs producing strains and MC-LR equivalence. Coliform bacteria, COD, NO3-N, and phosphate are the significant contaminated parameters favoring the growth of putative MCs producing cyanobacteria. National Sanitation Foundation WQI (NSFWQI) indicates water quality, either bad or medium category at sampling points. The morphological analysis confirms the occurrence of diverse cyanobacterial genera such as Microcystis, Anabaena, Oscillatoria, and Phormidium. PCR amplification affirmed the presence of toxic microcystin (mcy) genes in uncultured cyanobacteria at all the sampling sites. The concentration of MC-LR equivalence in water samples by protein phosphatase 1 inhibition assay (PPIA) and high-performance liquid chromatography (HPLC) methods was observed in the range of 23.4-172 ng/L and 13.2-97.5 ng/L respectively which is lower than the harmful exposure limit by World Health Organization (WHO). Ganga isolate 1 was identified as Microcystis based on partial 16S rDNA sequence and its toxicity was confirmed due to presence of mcy genes and MCs production potential. These findings suggest the presence of MCs producers as new emerging parameter to monitor water quality index and identification up to species level will be valuable for restoration strategies of river Ganga.

At the Intersection of Chemistry, Biology, and Medicine.

After an undergraduate degree in biology at Harvard, I started graduate school at The Rockefeller Institute for Medical Research in New York City in July 1965. I was attracted to the chemical side of biochemistry and joined Fritz Lipmann's large, hierarchical laboratory to study enzyme mechanisms. That work led to postdoctoral research with Robert Abeles at Brandeis, then a center of what, 30 years later, would be called chemical biology. I spent 15 years on the Massachusetts Institute of Technology faculty, in both the Chemistry and Biology Departments, and then 26 years on the Harvard Medical School Faculty. My research interests have been at the intersection of chemistry, biology, and medicine. One unanticipated major focus has been investigating the chemical logic and enzymatic machinery of natural product biosynthesis, including antibiotics and antitumor agents. In this postgenomic era it is now recognized that there may be from 105 to 106 biosynthetic gene clusters as yet uncharacterized for potential new therapeutic agents.

Handmade microfluidic device for biochemical applications in emulsion.

A simple, inexpensive flow-focusing device has been developed to make uniform droplets for biochemical reactions, such as in vitro transcription and cell-free protein synthesis. The device was fabricated from commercially available components without special equipment. Using the emulsion droplets formed by the device, a class I ligase ribozyme, bcI 23, was successfully synthesized from DNA attached to magnetic microbeads by T7 RNA polymerase. It was also ligated with an RNA substrate on the same microbeads, and detected using flow cytometry with a fluorescent probe. In addition, a single-chain derivative of the lambda Cro protein was expressed using an Escherichia coli cell-free protein synthesis system in emulsion, which was prepared using the flow-focusing device. In both emulsified reactions, usage of the flow-focusing device was able to greatly reduce the coefficient of variation for the amount of RNA or protein displayed on the microbeads, demonstrating the device is advantageous for quantitative analysis in high-throughput screening.

Pooled-DNA Sequencing for Elucidating New Genomic Risk Factors, Rare Variants Underlying Alzheimer's Disease.

Analyses of genome-wide association studies (GWAS) for complex disorders usually identify common variants with a relatively small effect size that only explain a small proportion of phenotypic heritability. Several studies have suggested that a significant fraction of heritability may be explained by low-frequency (minor allele frequency (MAF) of 1-5 %) and rare-variants that are not contained in the commercial GWAS genotyping arrays (Schork et al., Curr Opin Genet Dev 19:212, 2009). Rare variants can also have relatively large effects on risk for developing human diseases or disease phenotype (Cruchaga et al., PLoS One 7:e31039, 2012). However, it is necessary to perform next-generation sequencing (NGS) studies in a large population (>4,000 samples) to detect a significant rare-variant association. Several NGS methods, such as custom capture sequencing and amplicon-based sequencing, are designed to screen a small proportion of the genome, but most of these methods are limited in the number of samples that can be multiplexed (i.e. most sequencing kits only provide 96 distinct index). Additionally, the sequencing library preparation for 4,000 samples remains expensive and thus conducting NGS studies with the aforementioned methods are not feasible for most research laboratories.The need for low-cost large scale rare-variant detection makes pooled-DNA sequencing an ideally efficient and cost-effective technique to identify rare variants in target regions by sequencing hundreds to thousands of samples. Our recent work has demonstrated that pooled-DNA sequencing can accurately detect rare variants in targeted regions in multiple DNA samples with high sensitivity and specificity (Jin et al., Alzheimers Res Ther 4:34, 2012). In these studies we used a well-established pooled-DNA sequencing approach and a computational package, SPLINTER (short indel prediction by large deviation inference and nonlinear true frequency estimation by recursion) (Vallania et al., Genome Res 20:1711, 2010), for accurate identification of rare variants in large DNA pools. Given an average sequencing coverage of 30× per haploid genome, SPLINTER can detect rare variants and short indels up to 4 base pairs (bp) with high sensitivity and specificity (up to 1 haploid allele in a pool as large as 500 individuals). Step-by-step instructions on how to conduct pooled-DNA sequencing experiments and data analyses are described in this chapter.

A price to pay for relaxed substrate specificity: a comparative kinetic analysis of the class II lanthipeptide synthetases ProcM and HalM2.

Lanthipeptides are a class of ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) that typically harbor multiple intramolecular thioether linkages. For class II lanthipeptides, these cross-links are installed in a multistep reaction pathway by a single enzyme (LanM). The multifunctional nature of LanMs and the manipulability of their genetically encoded peptide substrates (LanAs) make LanM/LanA systems promising targets for the engineering of new antibacterial compounds. Here, we report the development of a semiquantitative mass spectrometry-based assay for kinetic characterization of LanM-catalyzed reactions. The assay was used to conduct a comparative kinetic analysis of two LanM enzymes (HalM2 and ProcM) that exhibit drastically different substrate selectivity. Numerical simulation of the kinetic data was used to develop models for the multistep HalM2- and ProcM-catalyzed reactions. These models illustrate that HalM2 and ProcM have markedly different catalytic efficiencies for the various reactions they catalyze. HalM2, which is responsible for the biosynthesis of a single compound (the Halß subunit of the lantibiotic haloduracin), catalyzes reactions with higher catalytic efficiency than ProcM, which modifies 29 different ProcA precursor peptides during prochlorosin biosynthesis. In particular, the rates of thioether ring formation are drastically reduced in ProcM, likely because this enzyme is charged with installing a variety of lanthipeptide ring architectures in its prochlorosin products. Thus, ProcM appears to pay a kinetic price for its relaxed substrate specificity. In addition, our kinetic models suggest that conformational sampling of the LanM/LanA Michaelis complex could play an important role in the kinetics of LanA maturation.

A ferrofluid-based homogeneous assay for highly sensitive and selective detection of single-nucleotide polymorphisms.

A simple and low-cost colorimetric assay utilizing ferrofluidic nanoparticulate probes (FNPs) and a ligase for single-nucleotide polymorphism genotyping is described. Excellent sensitivity and selectivity were accomplished through the engagement of the FNPs and a ligase chain reaction.

Optimization of a ligase detection reaction-fluorescent microsphere assay for characterization of resistance-mediating polymorphisms in African samples of Plasmodium falciparum.

Genetic polymorphisms in the malaria parasite Plasmodium falciparum mediate alterations in sensitivity to important antimalarial drugs. Surveillance for these polymorphisms is helpful in assessing the prevalence of drug resistance and designing strategies for malaria control. Multiple methods are available for the assessment of P. falciparum genetic polymorphisms, but they suffer from low throughput, technical limitations, and high cost. We have optimized and tested a multiplex ligase detection reaction-fluorescent microsphere (LDR-FM) assay for the identification of important P. falciparum genetic polymorphisms. For 84 clinical samples from Kampala, Uganda, a region where both transmission intensity and infection complexity are high, DNA was extracted from dried blood spots, genes of interest were amplified, amplicons were subjected to multiplex ligase detection reactions to add bead-specific oligonucleotides and biotin, fragments were hybridized to magnetic beads, and polymorphism prevalences were assessed fluorometrically in a multiplex format. A total of 19 alleles from the pfcrt, pfmdr1, pfmrp1, pfdhfr, and pfdhps genes were analyzed by LDR-FM and restriction fragment length polymorphism (RFLP) analyses. Considering samples with results from the two assays, concordance between the assays was good, with 78 to 100% of results identical at individual alleles, most nonconcordant results differing only between a mixed and pure genotype call, and full disagreement at individual alleles in only 0 to 3% of results. We estimate that the LDR-FM assay offers much higher throughput and lower cost than RFLP. Our results suggest that the LDR-FM system offers an accurate high-throughput means of classifying genetic polymorphisms in field samples of P. falciparum.

Evolution of lanthipeptide synthetases.

Lanthionine-containing peptides (lanthipeptides) are a family of ribosomally synthesized and posttranslationally modified peptides containing (methyl)lanthionine residues. Here we present a phylogenomic study of the four currently known classes of lanthipeptide synthetases (LanB and LanC for class I, LanM for class II, LanKC for class III, and LanL for class IV). Although they possess very similar cyclase domains, class II-IV synthetases have evolved independently, and LanB and LanC enzymes appear to not always have coevolved. LanM enzymes from various phyla that have three cysteines ligated to a zinc ion (as opposed to the more common Cys-Cys-His ligand set) cluster together. Most importantly, the phylogenomic data suggest that for some scaffolds, the ring topology of the final lanthipeptides may be determined in part by the sequence of the precursor peptides and not just by the biosynthetic enzymes. This notion was supported by studies with two chimeric peptides, suggesting that the nisin and prochlorosin biosynthetic enzymes can produce the correct ring topologies of epilancin 15X and lacticin 481, respectively. These results highlight the potential of lanthipeptide synthetases for bioengineering and combinatorial biosynthesis. Our study also demonstrates unexplored areas of sequence space that may be fruitful for genome mining.

The impact of structural biology on alkaloid biosynthesis research.

In the recent past, macromolecular crystallography has gone through substantial methodological and technological development. The purpose of this review is to provide a general overview of structural biology and its impact on enzyme structure/function analysis and illustrate how it is modifying the focus of research relevant to alkaloid biosynthesis.

Application of real-time PCR in the assessment of the toxic cyanobacterium Cylindrospermopsis raciborskii abundance and toxicological potential.

Cyanobacteria are prokaryotic photosynthetic microorganisms that pose a serious threat to aquatic environments because they are able to form blooms under eutrophic conditions and produce toxins. Cylindrospermopsis raciborskii is a planktonic heterocystous filamentous cyanobacterium initially assigned to the tropics but currently being found in more temperate regions such as Portugal, the southernmost record for this species in Europe. Cylindrospermopsin originally isolated from C. raciborskii is a cytotoxic alkaloid that affects the liver, kidney, and other organs. It has a great environmental impact associated with cattle mortality and human morbidity. Aiming in monitoring this cyanobacterium and its related toxin, a shallow pond located in the littoral center of Portugal, Vela Lake, used for agriculture and recreational purposes was monitored for a 2-year period. To accomplish this, we used the real-time PCR methodology in field samples to quantify the variation of specific genetic markers with primers previously described characterizing total cyanobacteria (16S rRNA), C. raciborskii (rpoC1), and cylindrospermopsin synthetase gene (pks). The results report the high abundance of both cyanobacteria and C. raciborskii in Vela Lake, with C. raciborskii representing 0.4% to 58% of the total cyanobacteria population. Cylindrospermopsin synthetase gene was detected in one of the samples. We believe that with the approach developed in this study, it will be possible to monitor C. raciborskii population dynamics and seasonal variation, as well as the potential toxin production in other aquatic environments.

Molecular assessment of Plasmodium falciparum resistance to antimalarial drugs in Papua New Guinea using an extended ligase detection reaction fluorescent microsphere assay.

Surveillance for Plasmodium falciparum drug resistance mutations is becoming an established tool for assessing antimalarial treatment effectiveness. We used an extended version of a high-throughput post-PCR multiplexed ligase detection reaction fluorescent microsphere assay (LDR-FMA) to detect single-nucleotide P. falciparum drug resistance polymorphisms in 402 isolates from children in Papua New Guinea (PNG) participating in an antimalarial treatment trial. There was a fixation of P. falciparum crt (pfcrt) K76T, pfdhfr C59R and S108N, and pfmdr1 mutations (92%, 93%, 95%, and 91%, respectively). Multiple mutations were frequent. Eighty-eight percent of isolates possessed a quintuple mutation (underlined), SVMNT, NRNI, KAA, and YYSND, in codons 72 to 76 for pfcrt; 51, 59, 108, and 164 for pfdhfr; 540, 581, and 613 for pfdhps; and 86, 184, 1034, 1042, and 1246 for pfmdr1, and four of these carried the K540E pfdhps allele. The pfmdr1 D1246Y mutation was associated with PCR-corrected day 42 in vivo treatment failure in children allocated piperaquine-dihydroartemisinin (P = 0.004). Although the pfmdr1 NFSDD haplotype was found in only four isolates, it has been associated with artemether-lumefantrine treatment failure in Africa. LDR-FMA allows the large-scale assessment of resistance-associated single-nucleotide polymorphisms (SNPs). Our findings reflect previous heavy 4-aminoquinoline/sulfadoxine-pyrimethamine use in PNG. Since artemether-lumefantrine and piperaquine-dihydroartemisinin will become first- and second-line treatments, respectively, the monitoring of pfmdr1 SNPs appears to be a high priority.

A simple template-dependent ligase ribozyme as the RNA replicase emerging first in the RNA world.

The "RNA world" hypothesis has offered a framework for both experimental and theoretical work in the field of the origin of life. An important concern about the hypothesis is how the RNA world could originate. It has long been speculated that a template-dependent RNA synthetase ribozyme, which catalyzed its own replication (thus, an "RNA replicase"), should have emerged first. However, experimental searches for such a replicase have so far been unsuccessful. This is primarily because of the large sequence length of candidate ribozymes, which mainly work in a polymerase-like way. Here, we propose that the replicase that emerged first would be a simple template-dependent ligase ribozyme, which loosely binds to template RNA and has a relatively low efficiency of catalyzing the formation of phosphodiester bonds between adjacently aligned nucleotides or oligonucleotides. We conducted a computer simulation to support this proposal and considered the factors that might affect the emergence of the ribozyme based on the parameter analysis in the simulation. We conclude that (1) a template-dependent ligase may be more likely than a template-dependent polymerase as an early replicase in the emergence of RNA-based replication; (2) such a ligase ribozyme could emerge and be stable against parasites under a broad range of parameters in our model; (3) the conditions shown to favor the initial appearance of a template-dependent ligase ribozyme do not favor its spread.

Cost of unneeded proteins in E. coli is reduced after several generations in exponential growth.

When E. coli cells express unneeded protein, they grow more slowly. Such penalty to fitness associated with making proteins is called protein cost. Protein cost is an important component in the cost-benefit tradeoffs that underlie the evolution of protein circuits, but its origins are still poorly understood. Here, we ask how the protein cost varies during the exponential growth phase of E. coli. We find that cells growing exponentially following an upshift from overnight culture show a large cost when producing unneeded proteins. However, after several generations, while still in exponential growth, the cells enter a phase where cost is much reduced despite vigorous unneeded protein production. We find that this reduced-cost phase depends on the ppGpp system, which adjusts the amount of ribosomes in the cell and does not occur after a downshift from rich to poor medium. These findings suggest that protein cost is a transient phenomenon that happens upon an upshift in conditions and that cost is reduced when ribosomes and other cellular systems have increased to their appropriate steady-state level in the new condition.

Identification of novel L-amino acid alpha-ligases through Hidden Markov Model-based profile analysis.

L-Amino acid alpha-ligase (Lal), catalyzing the formation of alpha-dipeptides from unprotected L-amino acids in an ATP-dependent manner, is used in cost-effective fermentative production of dipeptides. We searched for novel Lals by in silico screening using Hidden Markov Model-based profile analysis, and identified five novel Lals that showed low similarity and different substrate specificity from known Lals.

Sumoylation of Smad4, the common Smad mediator of transforming growth factor-beta family signaling.

Transforming growth factor-beta (TGF-beta) and TGF-beta-related factors regulate cell growth, differentiation, and apoptosis, and play key roles in normal development and tumorigenesis. TGF-beta family-induced changes in gene expression are mediated by serine/threonine kinase receptors at the cell surface and Smads as intracellular effectors. Receptor-activated Smads combine with a common Smad4 to translocate into the nucleus where they cooperate with other transcription factors to activate or repress transcription. The activities of the receptor-activated Smads are controlled by post-translational modifications such as phosphorylation and ubiquitylation. Here we show that Smad4 is modified by sumoylation. Sumoylation of Smad4 was enhanced by the conjugating enzyme Ubc9 and members of the PIAS family of SUMO ligases. A major sumoylation site in Smad4 was localized to Lys-159 in its linker segment with an additional site at Lys-113 in the MH-1 domain. Increased sumoylation in the presence of the PIASy E3 ligase correlated with targeting of Smad4 to subnuclear speckles that contain SUMO-1 and PIASy. Replacement of lysines 159 and 113 by arginines or increased sumoylation enhanced the stability of Smad4, and transcription in mammalian cells and Xenopus embryos. These observations suggest a role for Smad4 sumoylation in the regulation of TGF-beta signaling through Smads.