Phase variation as a major mechanism of adaptation in Mycobacterium tuberculosis complex.

Phase variation induced by insertions and deletions (INDELs) in genomic homopolymeric tracts (HT) can silence and regulate genes in pathogenic bacteria, but this process is not characterized in MTBC (Mycobacterium tuberculosis complex) adaptation. We leverage 31,428 diverse clinical isolates to identify genomic regions including phase-variants under positive selection. Of 87,651 INDEL events that emerge repeatedly across the phylogeny, 12.4% are phase-variants within HTs (0.02% of the genome by length). We estimated the in-vitro frameshift rate in a neutral HT at 100× the neutral substitution rate at [Formula: see text] frameshifts/HT/year. Using neutral evolution simulations, we identified 4,098 substitutions and 45 phase-variants to be putatively adaptive to MTBC (P < 0.002). We experimentally confirm that a putatively adaptive phase-variant alters the expression of espA, a critical mediator of ESX-1-dependent virulence. Our evidence supports the hypothesis that phase variation in the ESX-1 system of MTBC can act as a toggle between antigenicity and survival in the host.

Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions.

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

Mutation of wbtJ, a N-formyltransferase involved in O-antigen synthesis, results in biofilm formation, phase variation and attenuation in Francisella tularensis.

Two clinically important subspecies, Francisella tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) are responsible for most tularaemia cases, but these isolates typically form a weak biofilm under in vitro conditions. Phase variation of the F. tularensis lipopolysaccharide (LPS) has been reported in these subspecies, but the role of variation is unclear as LPS is crucial for virulence. We previously demonstrated that a subpopulation of LPS variants can constitutively form a robust biofilm in vitro, but it is unclear whether virulence was affected. In this study, we show that biofilm-forming variants of both fully virulent F. tularensis subspecies were highly attenuated in the murine tularaemia model by multiple challenge routes. Genomic sequencing was performed on these strains, which revealed that all biofilm-forming variants contained a lesion within the wbtJ gene, a formyltransferase involved in O-antigen synthesis. A ΔwbtJ deletion mutant recapitulated the biofilm, O-antigen and virulence phenotypes observed in natural variants and could be rescued through complementation with a functional wbtJ gene. Since the spontaneously derived biofilm-forming isolates in this study were a subpopulation of natural variants, reversion events to the wbtJ gene were detected that eliminated the phenotypes associated with biofilm variants and restored virulence. These results demonstrate a role for WbtJ in biofilm formation, LPS variation and virulence of F. tularensis.

Coordinated modulation of multiple processes through phase variation of a c-di-GMP phosphodiesterase in Clostridioides difficile.

The opportunistic nosocomial pathogen Clostridioides difficile exhibits phenotypic heterogeneity through phase variation, a stochastic, reversible process that modulates expression. In C. difficile, multiple sequences in the genome undergo inversion through site-specific recombination. Two such loci lie upstream of pdcB and pdcC, which encode phosphodiesterases (PDEs) that degrade the signaling molecule c-di-GMP. Numerous phenotypes are influenced by c-di-GMP in C. difficile including cell and colony morphology, motility, colonization, and virulence. In this study, we aimed to assess whether PdcB phase varies, identify the mechanism of regulation, and determine the effects on intracellular c-di-GMP levels and regulated phenotypes. We found that expression of pdcB is heterogeneous and the orientation of the invertible sequence, or 'pdcB switch', determines expression. The pdcB switch contains a promoter that when properly oriented promotes pdcB expression. Expression is augmented by an additional promoter upstream of the pdcB switch. Mutation of nucleotides at the site of recombination resulted in phase-locked strains with significant differences in pdcB expression. Characterization of these mutants showed that the pdcB locked-ON mutant has reduced intracellular c-di-GMP compared to the locked-OFF mutant, consistent with increased and decreased PdcB activity, respectively. These alterations in c-di-GMP had concomitant effects on multiple known c-di-GMP regulated processes, indicating that phase variation of PdcB allows C. difficile to coordinately diversify multiple phenotypes in the population to enhance survival.

The phase variation between wrinkly and smooth colony phenotype affects the virulence of Vibrio parahaemolyticus.

Vibrio parahaemolyticus, the causative agent of seafood-associated gastroenteritis, undergoes wrinkly and smooth colony switching on the plate. The wrinkly spreader grew faster, had stronger motility and biofilm capacity when compared with the smooth one. However, whether the two phenotypes differ in their virulence still needs to be further investigated. In this study, the data showed that the smooth spreader had stronger virulence phenotypes, including the cytotoxicity against HeLa cells, antibacterial activity against E. coli, adhesive capacity toward HeLa cells, and lethality in zebrafish, relative to the wrinkly one. However, the colony morphology variation had no influence on the haemolytic activity. The mRNA levels of major virulence genes including T3SS1, T6SS1, and T6SS2 were significantly enhanced in the smooth colonies relative to those in the wrinkly colonies. Taken together, the presented work highlighted the different virulence profiles of the wrinkly and smooth colony phenotypes.

Partial Fourier reconstruction of complex MR images using complex-valued convolutional neural networks.

PURPOSE: To provide a complex-valued deep learning approach for partial Fourier (PF) reconstruction of complex MR images. METHODS: Conventional PF reconstruction methods, such as projection onto convex sets (POCS), uses low-resolution image phase information from the central symmetrically sampled k-space for image reconstruction. However, this smooth phase constraint undermines the phase estimation accuracy in presence of rapid local phase variations, causing image artifacts and limiting the extent of PF reconstruction. Using both magnitude and phase characteristics in big complex image datasets, we propose a complex-valued deep learning approach with an unrolled network architecture for PF reconstruction that iteratively reconstructs PF sampled data and enforces data consistency. We evaluate our approach for reconstructing both spin-echo and gradient-echo data. RESULTS: The proposed method outperformed the iterative POCS PF reconstruction method. It produced better artifact suppression and recovery of both image magnitude and phase details in presence of local phase changes. No noise amplification was observed even for highly PF reconstruction. Moreover, the network trained on axial brain data could reconstruct sagittal and coronal brain and knee data. This method could be extended to 2D PF reconstruction and joint multi-slice PF reconstruction. CONCLUSION: Our proposed method can effectively reconstruct MR data even at low PF fractions, yielding high-fidelity magnitude and phase images. It presents a valuable alternative to conventional PF reconstruction, especially for phase-sensitive 2D or 3D MRI applications.

Dynamic mechanism of phase variation in bacteria based on multistable gene regulatory networks.

Phase variation, which causes splitting the homogeneous bacterial population into subpopulations of the cells with different and reversible activity of the same variable genes, provide bacteria with wide opportunities to implement their adaptation strategies. Meanwhile, the gene networks perform integral regulation of the genome and control over the formation of phenotypic traits of the organism. Mechanisms of phase variation, such as genomic rearrangements and DNA methylation, affect the regulatory contours of gene networks. At the same time, the gene networks themselves may be the key cause of the appearance of phenotypic variants. Here we propose a new class of gene networks that represent a ring connection of genetic triggers based on an oscillator with cyclic gene repression, termed the tringers. It is shown in silico that the tringers are able to provide splitting the homogeneous bacterial population into cellular subpopulations with alternative expression of controlled genes (phases), stable inheritance of phases, and switching between them, in particular, returning to a homogeneous state. Switching can be carried out under the influence of external and internal factors, both directly and by signals from specialized molecular genetic systems of the cell, in particular, the response to various types of stress. Such epigenetic structures with the function of a phase switching controller are possible in nature and applicable in synthetic biology.

Diversity in the swimming motility and flagellar regulon structure of uropathogenic Morganella morganii strains.

In current times, the opportunistic pathogen Morganella morganii is increasingly becoming a cause of urinary tract infections. The condition has been further complicated by the multiple drug resistance of most isolates. Swimming motility plays an important role in the development of urinary tract infections, allowing bacteria to colonize the upper urinary tract. We determined the differences between the growth, swimming motility, and biofilm formation of two M. morganii strains MM 1 and MM 190 isolated from the urine of patients who had community-acquired urinary tract infections. MM 190 showed a lower growth rate but better-formed biofilms in comparison to MM 1. In addition, MM 190 possessed autoaggregation abilities. It was found that a high temperature (37 °C) inhibits the flagellation of strains and makes MM 190 less motile. At the same time, the MM 1 strain maintained its rate of motility at this temperature. We demonstrated that urea at a concentration of 1.5% suppresses the growth and swimming motility of both strains. Genome analysis showed that MM 1 has a 17.7-kb-long insertion in flagellar regulon between fliE and glycosyltransferase genes, which was not identified in corresponding loci of MM 190 and 9 other M. morganii strains with whole genomes. Both strains carry two genes encoding flagellin, which may indicate flagellar antigen phase variation. However, the fliC2 genes have only 91% identity to each other and exhibit some variability in the regulatory region. We assume that all these differences influence the swimming motility of the strains.

Long-term persistence of crAss-like phage crAss001 is associated with phase variation in Bacteroides intestinalis.

BACKGROUND: The crAss-like phages are ubiquitous and highly abundant members of the human gut virome that infect commensal bacteria of the order Bacteroidales. Although incapable of lysogeny, these viruses demonstrate long-term persistence in the human gut microbiome, dominating the virome in some individuals. RESULTS: Here we show that rapid phase variation of alternate capsular polysaccharides in Bacteroides intestinalis cultures plays an important role in a dynamic equilibrium between phage sensitivity and resistance, allowing phage and bacteria to multiply in parallel. The data also suggests the role of a concomitant phage persistence mechanism associated with delayed lysis of infected cells, similar to carrier state infection. From an ecological and evolutionary standpoint, this type of phage-host interaction is consistent with the Piggyback-the-Winner model, which suggests a preference towards lysogenic or other "benign" forms of phage infection when the host is stably present at high abundance. CONCLUSION: Long-term persistence of bacteriophage and host could result from mutually beneficial mechanisms driving bacterial strain-level diversity and phage survival in complex environments.

Salmonella Regulator STM0347 Mediates Flagellar Phase Variation via Hin Invertase.

Salmonella enterica is one of the most important food-borne pathogens, whose motility and virulence are highly related to flagella. Flagella alternatively express two kinds of surface antigen flagellin, FliC and FljB, in a phenomenon known as flagellar phase variation. The molecular mechanisms by which the switching orientation of the Hin-composed DNA segment mediates the expression of the fljBA promoter have been thoroughly illustrated. However, the precise regulators that control DNA strand exchange are barely understood. In this study, we found that a putative response regulator, STM0347, contributed to the phase variation of flagellin in S. Typhimurium. With quantitative proteomics and secretome profiling, a lack of STM0347 was confirmed to induce the transformation of flagellin from FliC to FljB. Real-time PCR and in vitro incubation of SMT0347 with the hin DNA segment suggested that STM0347 disturbed Hin-catalyzed DNA reversion via hin degradation, and the overexpression of Hin was sufficient to elicit flagellin variation. Subsequently, the Δstm0347 strain was outcompeted by its parental strain in HeLa cell invasion. Collectively, our results reveal the crucial role of STM0347 in Salmonella virulence and flagellar phase variation and highlight the complexity of the regulatory network of Hin-modulated flagellum phase variation in Salmonella.

Deep learning based multiplexed sensitivity-encoding (DL-MUSE) for high-resolution multi-shot DWI.

PURPOSE: A phase correction method for high-resolution multi-shot (MSH) diffusion weighted imaging (DWI) is proposed. The efficacy and generalization capability of the method were validated on both healthy volunteers and patients. THEORY AND METHODS: Conventionally, inter-shot phase variations for MSH echo-planar imaging (EPI) DWI are corrected by model-based algorithms. However, many acquisition imperfections are hard to measure accurately for conventional model-based methods, making the phase estimation and artifacts suppression unreliable. We propose a deep learning multiplexed sensitivity-encoding (DL-MUSE) framework to improve the phase estimations based on convolutional neural network (CNN) reconstruction. Aliasing-free single-shot (SSH) DW images, which have been used routinely in clinical settings, were used for training before the aliasing correction of MSH-DWI images. A dual-channel U-net comprising multiple convolutional layers was used for the phase estimation of MSH-EPI. The network was trained on a dataset containing 30 healthy volunteers and tested on another dataset of 52 healthy subjects and 15 patients with lesions or tumors with different shot numbers (4, 6 and 8). To further validate the generalization capability of our network, we acquired a dataset with different numbers of shots, TEs, partial Fourier factors, resolutions, ETLs, FOVs, coil numbers, and image orientations from two sites. We also compared the reconstruction performance of our proposed method with that of the conventional MUSE and SSH-EPI qualitatively and quantitatively. RESULTS: Our results show that DL-MUSE is capable of correcting inter-shot phase errors with high and robust performance. Compared to conventional model-based MUSE, our method, by applying deep learning-based phase corrections, showed reduced distortion, noise level, and signal loss in high b-value DWIs. The improvements of image quality become more evident as the shot number increases from 4 to 8, especially in those central regions of the images, where g-factor artifacts are severe. Furthermore, the proposed method could provide the information about the orientation of the white matter with better consistency and achieve finer fibers delineation compared to the SSH-EPI method. Besides, the experiments on volunteers and patients from two different sites demonstrated the generalizability of our proposed method preliminarily. CONCLUSION: A deep learning-based reconstruction algorithm for MSH-EPI images, which helps improve image quality greatly, was proposed. Results from healthy volunteers and tumor patients demonstrated the feasibility and generalization performances of our method for high-resolution MSH-EPI DWI, which can be used for routine clinical applications as well as neuroimaging research.

Bacteria autoaggregation: how and why bacteria stick together.

Autoaggregation, adherence between identical bacterial cells, is important for colonization, kin and kind recognition, and survival of bacteria. It is directly mediated by specific interactions between proteins or organelles on the surfaces of interacting cells or indirectly by the presence of secreted macromolecules such as eDNA and exopolysaccharides. Some autoaggregation effectors are self-associating and present interesting paradigms for protein interaction. Autoaggregation can be beneficial or deleterious at specific times and niches. It is, therefore, typically regulated through transcriptional or post-transcriptional mechanisms or epigenetically by phase variation. Autoaggregation can contribute to bacterial adherence, biofilm formation or other higher-level functions. However, autoaggregation is only required for these phenotypes in some bacteria. Thus, autoaggregation should be detected, studied and measured independently using both qualitative and quantitative in vitro and ex vivo methods. If better understood, autoaggregation holds the potential for the discovery of new therapeutic targets that could be cost-effectively exploited.

Clinical features relating to pneumococcal colony phase variation in hospitalized adults with pneumonia.

Background. Streptococcus pneumoniae is a major causative bacteria of pneumonia and invasive pneumococcal disease (IPD); however, the mechanisms underlying its severity and invasion remain to be defined. Pneumococcal colonies exhibit opaque and transparent opacity phase variations, which have been associated with invasive infections and nasal colonization, respectively, in animal studies. This study evaluated the relationship between the opacity of pneumococcal colonies and the clinical presentation of pneumococcal pneumonia.Methods. This retrospective study included adult patients hospitalized with pneumococcal pneumonia between 2012 and 2019 at four tertiary medical institutions. Pneumococcal strains from lower respiratory tract specimens were determined for their serotypes and microscopic colony opacity, and the association between the opacity phase and the severity of pneumonia was evaluated. Serotypes 3 and 37 with mucoid colony phenotypes were excluded from the study because their colony morphologies were clearly different.Results. A total of 92 patients were included. Most patients were older adults (median age: 72 years) and males (67 %), and 59 % had community-acquired pneumonia. Of the 92 patients, 41 (45 %), 12 (13 %), and 39 (42 %) patients had opaque, transparent, and mixed variants in their pneumococcal colony, respectively. The opaque and non-opaque pneumococcal variants had no statistically significant difference in patient backgrounds. Although the pneumonia severity index score did not differ between the opaque and non-opaque groups, the rate of bacteremia was significantly higher in the opaque group than in the non-opaque group. Serotype distribution was similar between the groups.Conclusions. Opaque pneumococcal variants may cause pneumonia and invasive diseases in humans. This study could help elucidate IPD, and opacity assessment may serve as a predictor for IPD.

Phasevarions in Haemophilus influenzae biogroup aegyptius control expression of multiple proteins.

IMPORTANCE: Haemophilus influenzae biogroup aegyptius is a human-adapted pathogen and the causative agent of Brazilian purpuric fever (BPF), an invasive disease with high mortality, that sporadically manifests in children previously suffering conjunctivitis. Phase variation is a rapid and reversible switching of gene expression found in many bacterial species, and typically associated with outer-membrane proteins. Phase variation of cytoplasmic DNA methyltransferases has been shown to play important roles in bacterial gene regulation and can act as epigenetic switches, regulating the expression of multiple genes as part of systems called phasevarions (phase-variable regulons). This study characterized two alleles of the ModA phasevarion present in H. influenzae biogroup aegyptius, ModA13, found in non-BPF causing strains and ModA16, unique to BPF causing isolates. Phase variation of ModA13 and ModA16 led to genome-wide changes to DNA methylation resulting in altered protein expression. These changes did not affect serum resistance in H. influenzae biogroup aegyptius strains.

Phase Variation of Flagella and Toxins in Clostridioides difficile is Mediated by Selective Rho-dependent Termination.

Clostridioides difficile is an intestinal pathogen that exhibits phase variation of flagella and toxins through inversion of the flagellar (flg) switch controlling flagellar and toxin gene expression. The transcription termination factor Rho preferentially inhibits swimming motility of bacteria with the 'flg-OFF' switch sequence. How C. difficile Rho mediates this selectivity was unknown. C. difficile Rho contains an N-terminal insertion domain (NID) which is found in a subset of Rho orthologues and confers diverse functions. Here we determined how Rho distinguishes between flg-ON and -OFF mRNAs and the roles of the NID and other domains of C. difficile Rho. Using in vitro ATPase assays, we determined that Rho specifically binds a region containing the left inverted repeat of the flg switch, but only of flg-OFF mRNA, indicating that differential termination is mediated by selective Rho binding. Using a suite of in vivo and in vitro assays in C. difficile, we determined that the NID is essential for Rho termination of flg-OFF mRNA, likely by influencing the ability to form stable hexamers, and the RNA binding domain is critical for flg-OFF specific termination. This work gives insight into the novel mechanism by which Rho interacts with flg mRNA to mediate phase variation of flagella and toxins in C. difficile and broadens our understanding of Rho-mediated termination in an organism with an AT-rich genome.

Colony phase variation switch modulates antimicrobial tolerance and biofilm formation in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii causes one of the most difficult-to-treat nosocomial infections. Polycationic drugs like polymyxin B or colistin and tetracycline drugs such as doxycycline or minocycline are commonly used to treat infections caused by carbapenem-resistant A. baumannii. Here, we show that a subpopulation of cells associated with the opaque/translucent colony phase variation by A. baumannii AB5075 displays differential tolerance to subinhibitory concentrations of colistin and tetracycline. Using a variety of microscopic techniques, we demonstrate that extracellular polysaccharide moieties mediate colistin tolerance to opaque A. baumannii at single-cell level and that mushroom-shaped biofilm structures protect opaque bacteria at the community level. The colony switch phenotype is found to alter several traits of A. baumannii, including long-term survival under desiccation, tolerance to ethanol, competition with Escherichia coli, and intracellular survival in the environmental model host Acanthamoeba castellanii. Additionally, our findings suggest that extracellular DNA associated with membrane vesicles can promote colony switching in a DNA recombinase-dependent manner.IMPORTANCEAs a WHO top-priority drug-resistant microbe, Acinetobacter baumannii significantly contributes to hospital-associated infections worldwide. One particularly intriguing aspect is its ability to reversibly switch its colony morphotype on agar plates, which has been remarkably underexplored. In this study, we employed various microscopic techniques and phenotypic assays to investigate the colony phase variation switch under different clinically and environmentally relevant conditions. Our findings reveal that the presence of a poly N-acetylglucosamine-positive extracellular matrix layer contributes to the protection of bacteria from the bactericidal effects of colistin. Furthermore, we provide intriguing insights into the multicellular lifestyle of A. baumannii, specifically in the context of colony switch variation within its predatory host, Acanthamoeba castellanii.

Adherence of Nontypeable Haemophilus influenzae to Cells and Substrates of the Airway Is Differentially Regulated by Individual ModA Phasevarions.

Adherence of nontypeable Haemophilus influenzae (NTHi) to the host airway is an essential initial step for asymptomatic colonization of the nasopharynx, as well as development of disease. NTHi relies on strict regulation of multiple adhesins for adherence to host substrates encountered in the airway. NTHi encode a phase-variable cytoplasmic DNA methyltransferase, ModA, that regulates expression of multiple genes; a phasevarion (phase-variable regulon). Multiple modA alleles are present in NTHi, in which different alleles methylate a different DNA target, and each controls a different set of genes. However, the role of ModA phasevarions in regulating adherence of NTHi to the host airway is not well understood. This study therefore sought to investigate the role of four of the most prevalent ModA phasevarions in the regulation of adherence of NTHi to multiple substrates of the airway. Four clinical isolates of NTHi with unique modA alleles were tested in this study. The adherence of NTHi to mucus, middle ear epithelial cells, and vitronectin was regulated in a substrate-specific manner that was dependent on the ModA allele encoded. The adhesins Protein E and P4 were found to contribute to the ModA-regulated adherence of NTHi to distinct substrates. A better understanding of substrate-specific regulation of NTHi adherence by ModA phasevarions will allow identification of NTHi populations present at the site of disease within the airway and facilitate more directed development of vaccines and therapeutics. IMPORTANCE Nontypeable Haemophilus influenzae (NTHi) is a predominant pathogen of the human airway that causes respiratory infections such as otitis media (OM) and exacerbations in the lungs of patients suffering from chronic obstructive pulmonary disease (COPD). Due to the lack of a licensed vaccine against NTHi and the emergence of antibiotic-resistant strains, it is extremely challenging to target NTHi for treatment. NTHi adhesins are considered potential candidates for vaccines or other therapeutic approaches. The ModA phasevarions of NTHi play a role in the rapid adaptation of the pathogen to different environmental stress conditions. This study addressed the role of ModA phasevarions in the regulation of adherence of NTHi to specific host substrates found within the respiratory tract. The findings of this study improve our understanding of regulation of adherence of NTHi to the airway, which may further be used to enhance the potential of adhesins as vaccine antigens and therapeutic targets against NTHi.

Phase variation of manganese oxide in the MnO@ZnO nanocomposite with calcination temperature and its effect on structural and biological activities.

Having powerful antibacterial and antioxidant effects, zinc oxide and manganese oxide nanomaterials are of great interest. Here we have synthesized manganese oxide decorated zinc oxide (MZO) nanocomposites by co-precipitation method, calcined at different temperatures (300-750 °C) and studied various properties. Here the crystalline structure of the nanocomposite and phase change of the manganese oxide are observed with calcination temperature. The average crystalline size increases and the dislocation density and microstrain decrease with the increase in calcined temperature for the same structural features. The formation of composites was confirmed by XRD pattern and SEM images. EDAX spectra proved the high purity of the composites. Here, different biological properties change with the calcination temperature for different shapes, sizes and structures of the nanocomposite. Nanomaterial calcined at 750 °C provides the best anti-microbial activity against Escherichia coli, Salmonella typhimurium, Shigella flexneri (gram-negative), Bacillus subtilis and Bacillus megaterium (gram-positive) bacterial strain at 300 µg/mL concentration. The nanomaterial with calcination temperatures of 300 °C and 450 °C provided better antioxidant properties.

Pneumococcal Phasevarions Control Multiple Virulence Traits, Including Vaccine Candidate Expression.

Streptococcus pneumoniae is the most common cause of bacterial illness worldwide. Current vaccines based on the polysaccharide capsule are only effective against a limited number of the >100 capsular serotypes. A universal vaccine based on conserved protein antigens requires a thorough understanding of gene expression in S. pneumoniae. All S. pneumoniae strains encode the SpnIII Restriction-Modification system. This system contains a phase-variable methyltransferase that switches specificity, and controls expression of multiple genes-a phasevarion. We examined the role of this phasevarion during pneumococcal pathobiology, and determined if phase variation resulted in differences in expression of currently investigated conserved protein antigens. Using locked strains that express a single methyltransferase specificity, we found differences in clinically relevant traits, including survival in blood, and adherence to and invasion of human cells. We also observed differences in expression of numerous proteinaceous vaccine candidates, which complicates selection of antigens for inclusion in a universal protein-based pneumococcal vaccine. This study will inform vaccine design against S. pneumoniae by ensuring only stably expressed candidates are included in a rationally designed vaccine. IMPORTANCE S. pneumoniae is the world's foremost bacterial pathogen. S. pneumoniae encodes a phasevarion (phase-variable regulon), that results in differential expression of multiple genes. Previous work demonstrated that the pneumococcal SpnIII phasevarion switches between six different expression states, generating six unique phenotypic variants in a pneumococcal population. Here, we show that this phasevarion generates multiple phenotypic differences relevant to pathobiology. Importantly, expression of conserved protein antigens varies with phasevarion switching. As capsule expression, a major pneumococcal virulence factor, is also controlled by the phasevarion, our work will inform the selection of the best candidates to include in a rationally designed, universal pneumococcal vaccine.

Distortion-Free Diffusion Imaging Using Self-Navigated Cartesian Echo-Planar Time Resolved Acquisition and Joint Magnitude and Phase Constrained Reconstruction.

Echo-planar time resolved imaging (EPTI) is an effective approach for acquiring high-quality distortion-free images with a multi-shot EPI (ms-EPI) readout. As with traditional ms-EPI acquisitions, inter-shot phase variations present a main challenge when incorporating EPTI into a diffusion-prepared pulse sequence. The aim of this study is to develop a self-navigated Cartesian EPTI-based (scEPTI) acquisition together with a magnitude and phase constrained reconstruction for distortion-free diffusion imaging. A self-navigated Cartesian EPTI-based diffusion-prepared pulse sequence is designed. The different phase components in EPTI diffusion signal are analyzed and an approach to synthesize a fully phase-matched navigator for the inter-shot phase correction is demonstrated. Lastly, EPTI contains richer magnitude and phase information than conventional ms-EPI, such as the magnitude and phase correlations along the temporal dimension. The potential of these magnitude and phase correlations to enhance the reconstruction is explored. The reconstruction results with and without phase matching and with and without phase or magnitude constraints are compared. Compared with reconstruction without phase matching, the proposed phase matching method can improve the accuracy of inter-shot phase correction and reduce signal corruption in the final diffusion images. Magnitude constraints further improve image quality by suppressing the background noise and thereby increasing SNR, while phase constraints can mitigate possible image blurring from adding magnitude constraints. The high-quality distortion-free diffusion images and simultaneous diffusion-relaxometry imaging capacity provided by the proposed EPTI design represent a highly valuable tool for both clinical and neuroscientific assessments of tissue microstructure.