Modeling of African population history using f-statistics is biased when applying all previously proposed SNP ascertainment schemes.

f-statistics have emerged as a first line of analysis for making inferences about demographic history from genome-wide data. Not only are they guaranteed to allow robust tests of the fits of proposed models of population history to data when analyzing full genome sequencing data-that is, all single nucleotide polymorphisms (SNPs) in the individuals being analyzed-but they are also guaranteed to allow robust tests of models for SNPs ascertained as polymorphic in a population that is an outgroup in a phylogenetic sense to all groups being analyzed. True "outgroup ascertainment" is in practice impossible in humans because our species has arisen from a substructured ancestral population that does not descend from a homogeneous ancestral population going back many hundreds of thousands of years into the past. However, initial studies suggested that non-outgroup-ascertainment schemes might produce robust enough results using f-statistics, and that motivated widespread fitting of models to data using non-outgroup-ascertained SNP panels such as the "Affymetrix Human Origins array" which has been genotyped on thousands of modern individuals from hundreds of populations, or the "1240k" in-solution enrichment reagent which has been the source of about 70% of published genome-wide data for ancient humans. In this study, we show that while analyses of population history using such panels work well for studies of relationships among non-African populations and one African outgroup, when co-modeling more than one sub-Saharan African and/or archaic human groups (Neanderthals and Denisovans), fitting of f-statistics to such SNP sets is expected to frequently lead to false rejection of true demographic histories, and failure to reject incorrect models. Analyzing panels of SNPs polymorphic in archaic humans, which has been suggested as a solution for the ascertainment problem, has limited statistical power and retains important biases. However, by carrying out simulations of diverse demographic histories, we show that bias in inferences based on f-statistics can be minimized by ascertaining on variants common in a union of diverse African groups; such ascertainment retains high statistical power while allowing co-analysis of archaic and modern groups.

Three assays for in-solution enrichment of ancient human DNA at more than a million SNPs.

The strategy of in-solution enrichment for hundreds of thousands of single-nucleotide polymorphisms (SNPs) has been used to analyze >70% of individuals with genome-scale ancient DNA published to date. This approach makes it economical to study ancient samples with low proportions of human DNA and increases the rate of conversion of sampled remains into interpretable data. So far, nearly all such data have been generated using a set of bait sequences targeting about 1.24 million SNPs (the "1240k reagent"), but synthesis of the reagent has been cost-effective for only a few laboratories. In 2021, two companies, Daicel Arbor Biosciences and Twist Bioscience, made available assays that target the same core set of SNPs along with supplementary content. We test all three assays on a common set of 27 ancient DNA libraries and show that all three are effective at enriching many hundreds of thousands of SNPs. For all assays, one round of enrichment produces data that are as useful as two. In our testing, the "Twist Ancient DNA" assay produces the highest coverages, greatest uniformity on targeted positions, and almost no bias toward enriching one allele more than another relative to shotgun sequencing. We also identify hundreds of thousands of targeted SNPs for which there is minimal allelic bias when comparing 1240k data to either shotgun or Twist data. This facilitates coanalysis of the large data sets that have been generated using 1240k and Twist capture, as well as shotgun sequencing approaches.

An integrative skeletal and paleogenomic analysis of stature variation suggests relatively reduced health for early European farmers.

Human culture, biology, and health were shaped dramatically by the onset of agriculture ∼12,000 y B.P. This shift is hypothesized to have resulted in increased individual fitness and population growth as evidenced by archaeological and population genomic data alongside a decline in physiological health as inferred from skeletal remains. Here, we consider osteological and ancient DNA data from the same prehistoric individuals to study human stature variation as a proxy for health across a transition to agriculture. Specifically, we compared "predicted" genetic contributions to height from paleogenomic data and "achieved" adult osteological height estimated from long bone measurements for 167 individuals across Europe spanning the Upper Paleolithic to Iron Age (∼38,000 to 2,400 B.P.). We found that individuals from the Neolithic were shorter than expected (given their individual polygenic height scores) by an average of −3.82 cm relative to individuals from the Upper Paleolithic and Mesolithic (P = 0.040) and −2.21 cm shorter relative to post-Neolithic individuals (P = 0.068), with osteological vs. expected stature steadily increasing across the Copper (+1.95 cm relative to the Neolithic), Bronze (+2.70 cm), and Iron (+3.27 cm) Ages. These results were attenuated when we additionally accounted for genome-wide genetic ancestry variation: for example, with Neolithic individuals −2.82 cm shorter than expected on average relative to pre-Neolithic individuals (P = 0.120). We also incorporated observations of paleopathological indicators of nonspecific stress that can persist from childhood to adulthood in skeletal remains into our model. Overall, our work highlights the potential of integrating disparate datasets to explore proxies of health in prehistory.

Transesophageal echocardiography-guided versus fluoroscopy-guided patent foramen ovale closure: A single center registry.

BACKGROUND: Percutaneous closure of patent foramen ovale (PFO) is conventionally performed under continuous transesophageal echocardiographic (TEE) guidance. We aimed to evaluate whether a simplified procedural approach, including pure fluoroscopy-guidance and final TEE control, as well as an aimed 'next-day-discharge' is comparable with the conventional TEE-guided procedure in terms of periprocedural and intermediate-term outcomes. METHODS: All patients who underwent a PFO closure at our center between 2010 and 2022 were retrospectively included. Prior to June 2019 cases were performed with continuous TEE guidance (TEE-guided group). Since June 2019, only pure fluoroscopy-guided PFO closures have been performed with TEE insertion and control just prior to device release (fluoroscopy-guided group). We analyzed procedural aspects, as well as long term clinical and echocardiographic outcomes. RESULTS: In total 291 patients were included in the analysis: 197 in the TEE-guided group and 94 in the fluoroscopy-guided group. Fluoroscopy-guided procedures were markedly shorter (48 ± 20 min vs. 25 ± 9 min; p < .01). There was no difference in procedural complications, including death, major bleeding, device dislodgement, stroke or clinically relevant peripheral embolization between the two groups (.5% vs. 0%; p = .99). Hospital stay was also shorter with the simplified approach (2.5 ± 1.6 vs. 3.5 ± 1.2 days; p < .01), allowing 85% same-day discharges during the last 12 months of observation period. At 6 ± 3 months echocardiographic follow-up a residual leakage was described in 8% of the TEE-guided cases and 2% of the fluoroscopy-guided cases (p = .08). CONCLUSION: While a complete TEE-free PFO closure might have potential procedural risks, our approach of pure fluoroscopy-guided with a brisk final TEE check seems to be advantageous in terms of procedural aspects with no sign of any acute or intermediate-term hazard and it could offer an equitable compromise between the two worlds: a complete TEE procedure and a procedure without any TEE.

A two-center pilot study on the effects of clinical ethics support on coercive measures in psychiatry.

BACKGROUND: The use of formal coercion such as seclusion, mechanical restraint, and forced medication is one of the most challenging and complex issues in mental health care, on the clinical, the legal, and the ethical level. Clinical ethics support aims at assisting healthcare practitioners in determining the morally most justifiable course of action in these situations. However, the effectiveness of clinical ethics support has hardly been studied so far. METHODS: Monthly moral case deliberation (MCD) was implemented in two acute wards of two different psychiatric hospitals in Switzerland. Frequency and intensity of coercion was measured on ward level (npatients = 405), and the Moral Attentiveness Scale, Knowledge on Coercion Scale, and Staff Attitudes towards Coercion Scale were applied on healthcare practitioner level (nHP = 46). Pre-post-comparisons were conducted using multi-level modeling where appropriate. RESULTS: After implementation of MCD, formal coercion was less frequent (particularly seclusion, small effect size; 9.6 vs. 16.7%, p = .034, Cramér's V = .105) and less intense (particularly mechanical restraint, large effect size; 86.8 ± 45.3 vs. 14.5 ± 12.1 h, exact p = .019, r = -.74), and approval for coercive measures among healthcare practitioners was lower when controlling for the number of MCD sessions attended. CONCLUSIONS: Clinical ethics support such as MCD may be a hitherto underutilized service for the reduction of coercion, complementing existing strategies and programs. Implementing clinical ethics support may help improve quality of care for persons suffering from severe mental illness.

Genetic contributions to variation in human stature in prehistoric Europe.

The relative contributions of genetics and environment to temporal and geographic variation in human height remain largely unknown. Ancient DNA has identified changes in genetic ancestry over time, but it is not clear whether those changes in ancestry are associated with changes in height. Here, we directly test whether changes over the past 38,000 y in European height predicted using DNA from 1,071 ancient individuals are consistent with changes observed in 1,159 skeletal remains from comparable populations. We show that the observed decrease in height between the Early Upper Paleolithic and the Mesolithic is qualitatively predicted by genetics. Similarly, both skeletal and genetic height remained constant between the Mesolithic and Neolithic and increased between the Neolithic and Bronze Age. Sitting height changes much less than standing height-consistent with genetic predictions-although genetics predicts a small post-Neolithic increase that is not observed in skeletal remains. Geographic variation in stature is also qualitatively consistent with genetic predictions, particularly with respect to latitude. Finally, we hypothesize that an observed decrease in genetic heel bone mineral density in the Neolithic reflects adaptation to the decreased mobility indicated by decreased femoral bending strength. This study provides a model for interpreting phenotypic changes predicted from ancient DNA and demonstrates how they can be combined with phenotypic measurements to understand the relative contribution of genetic and developmentally plastic responses to environmental change.

Review of Microfluidic Devices and Imaging Techniques for Fluid Flow Study in Porous Geomaterials.

Understanding transport phenomena and governing mechanisms of different physical and chemical processes in porous media has been a critical research area for decades. Correlating fluid flow behaviour at the micro-scale with macro-scale parameters, such as relative permeability and capillary pressure, is key to understanding the processes governing subsurface systems, and this in turn allows us to improve the accuracy of modelling and simulations of transport phenomena at a large scale. Over the last two decades, there have been significant developments in our understanding of pore-scale processes and modelling of complex underground systems. Microfluidic devices (micromodels) and imaging techniques, as facilitators to link experimental observations to simulation, have greatly contributed to these achievements. Although several reviews exist covering separately advances in one of these two areas, we present here a detailed review integrating recent advances and applications in both micromodels and imaging techniques. This includes a comprehensive analysis of critical aspects of fabrication techniques of micromodels, and the most recent advances such as embedding fibre optic sensors in micromodels for research applications. To complete the analysis of visualization techniques, we have thoroughly reviewed the most applicable imaging techniques in the area of geoscience and geo-energy. Moreover, the integration of microfluidic devices and imaging techniques was highlighted as appropriate. In this review, we focus particularly on four prominent yet very wide application areas, namely "fluid flow in porous media", "flow in heterogeneous rocks and fractures", "reactive transport, solute and colloid transport", and finally "porous media characterization". In summary, this review provides an in-depth analysis of micromodels and imaging techniques that can help to guide future research in the in-situ visualization of fluid flow in porous media.

Iron-sulfur protein maturation in Helicobacter pylori: identifying a Nfu-type cluster carrier protein and its iron-sulfur protein targets.

Helicobacter pylori is anomalous among non nitrogen-fixing bacteria in containing an incomplete NIF system for Fe-S cluster assembly comprising two essential proteins, NifS (cysteine desulfurase) and NifU (scaffold protein). Although nifU deletion strains cannot be obtained via the conventional gene replacement, a NifU-depleted strain was constructed and shown to be more sensitive to oxidative stress compared to wild-type (WT) strains. The hp1492 gene, encoding a putative Nfu-type Fe-S cluster carrier protein, was disrupted in three different H. pylori strains, indicating that it is not essential. However, Δnfu strains have growth deficiency, are more sensitive to oxidative stress and are unable to colonize mouse stomachs. Moreover, Δnfu strains have lower aconitase activity but higher hydrogenase activity than the WT. Recombinant Nfu was found to bind either one [2Fe-2S] or [4Fe-4S] cluster/dimer, based on analytical, UV-visible absorption/CD and resonance Raman studies. A bacterial two-hybrid system was used to ascertain interactions between Nfu, NifS, NifU and each of 36 putative Fe-S-containing target proteins. Nfu, NifS and NifU were found to interact with 15, 6 and 29 putative Fe-S proteins respectively. The results indicate that Nfu, NifS and NifU play a major role in the biosynthesis and/or delivery of Fe-S clusters in H. pylori.

Quantifying between-cohort and between-sex genetic heterogeneity in major depressive disorder.

Major depressive disorder (MDD) is clinically heterogeneous with prevalence rates twice as high in women as in men. There are many possible sources of heterogeneity in MDD most of which are not measured in a sufficiently comparable way across study samples. Here, we assess genetic heterogeneity based on two fundamental measures, between-cohort and between-sex heterogeneity. First, we used genome-wide association study (GWAS) summary statistics to investigate between-cohort genetic heterogeneity using the 29 research cohorts of the Psychiatric Genomics Consortium (PGC; N cases = 16,823, N controls = 25,632) and found that some of the cohort heterogeneity can be attributed to ascertainment differences (such as recruitment of cases from hospital vs. community sources). Second, we evaluated between-sex genetic heterogeneity using GWAS summary statistics from the PGC, Kaiser Permanente GERA, UK Biobank, and the Danish iPSYCH studies but did not find convincing evidence for genetic differences between the sexes. We conclude that there is no evidence that the heterogeneity between MDD data sets and between sexes reflects genetic heterogeneity. Larger sample sizes with detailed phenotypic records and genomic data remain the key to overcome heterogeneity inherent in assessment of MDD.

Noncatalytic Antioxidant Role for Helicobacter pylori Urease.

The well-studied catalytic role of urease, the Ni-dependent conversion of urea into carbon dioxide and ammonia, has been shown to protect Helicobacter pylori against the low pH environment of the stomach lumen. We hypothesized that the abundantly expressed urease protein can play another noncatalytic role in combating oxidative stress via Met residue-mediated quenching of harmful oxidants. Three catalytically inactive urease mutant strains were constructed by single substitutions of Ni binding residues. The mutant versions synthesize normal levels of urease, and the altered versions retained all methionine residues. The three site-directed urease mutants were able to better withstand a hypochlorous acid (HOCl) challenge than a ΔureAB deletion strain. The capacity of purified urease to protect whole cells via oxidant quenching was assessed by adding urease enzyme to nongrowing HOCl-exposed cells. No wild-type cells were recovered with oxidant alone, whereas urease addition significantly aided viability. These results suggest that urease can protect H. pylori against oxidative damage and that the protective ability is distinct from the well-characterized catalytic role. To determine the capability of methionine sulfoxide reductase (Msr) to reduce oxidized Met residues in urease, purified H. pylori urease was exposed to HOCl and a previously described Msr peptide repair mixture was added. Of the 25 methionine residues in urease, 11 were subject to both oxidation and to Msr-mediated repair, as identified by mass spectrometry (MS) analysis; therefore, the oxidant-quenchable Met pool comprising urease can be recycled by the Msr repair system. Noncatalytic urease appears to play an important role in oxidant protection.IMPORTANCE Chronic Helicobacter pylori infection can lead to gastric ulcers and gastric cancers. The enzyme urease contributes to the survival of the bacterium in the harsh environment of the stomach by increasing the local pH. In addition to combating acid, H. pylori must survive host-produced reactive oxygen species to persist in the gastric mucosa. We describe a cyclic amino acid-based antioxidant role of urease, whereby oxidized methionine residues can be recycled by methionine sulfoxide reductase to again quench oxidants. This work expands our understanding of the role of an already acknowledged pathogen virulence factor and specifically expands our knowledge of H. pylori survival mechanisms.

Joint analysis of psychiatric disorders increases accuracy of risk prediction for schizophrenia, bipolar disorder, and major depressive disorder.

Genetic risk prediction has several potential applications in medical research and clinical practice and could be used, for example, to stratify a heterogeneous population of patients by their predicted genetic risk. However, for polygenic traits, such as psychiatric disorders, the accuracy of risk prediction is low. Here we use a multivariate linear mixed model and apply multi-trait genomic best linear unbiased prediction for genetic risk prediction. This method exploits correlations between disorders and simultaneously evaluates individual risk for each disorder. We show that the multivariate approach significantly increases the prediction accuracy for schizophrenia, bipolar disorder, and major depressive disorder in the discovery as well as in independent validation datasets. By grouping SNPs based on genome annotation and fitting multiple random effects, we show that the prediction accuracy could be further improved. The gain in prediction accuracy of the multivariate approach is equivalent to an increase in sample size of 34% for schizophrenia, 68% for bipolar disorder, and 76% for major depressive disorders using single trait models. Because our approach can be readily applied to any number of GWAS datasets of correlated traits, it is a flexible and powerful tool to maximize prediction accuracy. With current sample size, risk predictors are not useful in a clinical setting but already are a valuable research tool, for example in experimental designs comparing cases with high and low polygenic risk.

Helicobacter pylori nickel storage proteins: recognition and modulation of diverse metabolic targets.

Nickel metabolism and trafficking in Helicobacter pylori is complex, perhaps more so than in any other pathogen. Along with nickel enzymes and their associated nickel-binding maturation machinery, H. pylori contains nickel storage proteins, Hpn and Hpnl. Through a combined crosslinking and enrichment approach, we show that Hpn/Hpnl interact with a wide array of partners; over 100 proteins were captured, including known nickel-enzyme maturation proteins, and other proteins outside known H. pylori nickel-associated proteins. The crosslinker binds to exposed amines, but there was no correlation between lysine content and the pulldown abundance of captured proteins. Phenotypic characterization of mutant strains (Δhpn, Δhpnl, or ΔhpnΔhpnl) was used to explore interactions. Nickel deprivation affected the hydrogenase activity of the ΔhpnΔhpnl strain much more severely than the wild-type (WT), whereas the activities of the single mutants were similar to WT. Leucyl aminopeptidase activity was affected in opposite ways in the mutant strains: Δhpn had a threefold decrease, while Δhpnl had a sevenfold increase, compared to the parent. Similar mutant strain analysis supported Hpn and Hpnl acting synergistically to suppress aliphatic amidase activity in a nickel-dependent manner. Recombinant amidase could bind a variety of divalent metals. Amidase activity was greatest in the mutant strains and was inhibited by exogenous nickel. The addition of pure storage protein to extracts from the mutants only restored the suppression of amidase activity for the mutant strain lacking that protein; both storage proteins are needed for amidase suppression. These results suggest that Hpn and Hpnl play more diverse roles than previously thought.

Transport properties and size exclusion effects in wide-pore superficially porous particles.

The effects of hydrodynamic radius on the transport of solute molecules in packed beds of wide-pore superficially porous particles (SPP) are studied using pore-scale simulation. The free molecular diffusion rate varies with radius through the Stokes-Einstein relation. Lattice Boltzmann and Langevin methods are used to model fluid motion and the transport of an ensemble of solute molecules in the fluid, providing statistics on solute concentration, flux, molecule age and residence time, as a function of depth in the SPP. Intraparticle effective diffusion and bed dispersion coefficients are calculated and correlated with the hydrodynamic radius and accessible porosity. The relative importance of convection and diffusion are found to depend on the molecule (tracer) size through the diffusion rate, and convection effects are more significant for larger, slower-diffusing molecules. When larger molecules are utilized, the intraparticle concentration is reduced in proportion to the local particle porosity, leading to a natural definition of the accessible porosity used in size exclusion chromatography (SEC). Although the pore shape is complex, the SEC constant K can be calculated directly from simulation. Simulation demonstrates that the effective diffusion coefficient is elevated near the particle hull, which is largely open to interstitial flow, and decreases with depth into the particle. All molecules studied here have transport access to the entire particle depth, although the accessible volume at a given depth depends on their size. The first passage time into the particle is well predicted by the diffusion rate, but residence time is influenced by convection, shortening the average visit duration. These results are of interest in "perfusion" chromatography where convection is thought to increase separation efficiency for large biomolecules.

Helicobacter Catalase Devoid of Catalytic Activity Protects the Bacterium against Oxidative Stress.

Catalase, a conserved and abundant enzyme found in all domains of life, dissipates the oxidant hydrogen peroxide (H2O2). The gastric pathogen Helicobacter pylori undergoes host-mediated oxidant stress exposure, and its catalase contains oxidizable methionine (Met) residues. We hypothesized catalase may play a large stress-combating role independent of its classical catalytic one, namely quenching harmful oxidants through its recyclable Met residues, resulting in oxidant protection to the bacterium. Two Helicobacter mutant strains (katAH56A and katAY339A) containing catalase without enzyme activity but that retain all Met residues were created. These strains were much more resistant to oxidants than a catalase-deletion mutant strain. The quenching ability of the altered versions was shown, whereby oxidant-stressed (HOCl-exposed) Helicobacter retained viability even upon extracellular addition of the inactive versions of catalase, in contrast to cells receiving HOCl alone. The importance of the methionine-mediated quenching to the pathogen residing in the oxidant-rich gastric mucus was studied. In contrast to a catalase-null strain, both site-change mutants proficiently colonized the murine gastric mucosa, suggesting that the amino acid composition-dependent oxidant-quenching role of catalase is more important than the well described H2O2-dissipating catalytic role. Over 100 years after the discovery of catalase, these findings reveal a new non-enzymatic protective mechanism of action for the ubiquitous enzyme.

Atrial fibrillation in transcatheter aortic valve implantation patients: Incidence, outcome and predictors of new onset.

BACKGROUND: There is controversial evidence if atrial fibrillation (AF) alters outcome after transcatheter aortic valve implantation (TAVI). TAVI itself may promote new-onset AF (NOAF). METHODS: We performed a single-center study including 398 consecutive patients undergoing TAVI. Before TAVI, patients were divided into a sinus rhythm (SR) group (n=226, 57%) and baseline AF group (n=172, 43%) according to clinical records and electrocardiograms. Furthermore, incidence and predictors of NOAF were recorded. RESULTS: Baseline AF patients had a significantly higher 1-year mortality than the baseline SR group (19.8% vs. 11.5%, p=0.02). NOAF occurred in 7.1% of patients with prior SR. Previous valve surgery was the only significant predictor of NOAF (HR 5.86 [1.04-32.94], p<0.05). NOAF was associated with higher rehospitalization rate (62.5 vs. 34.8%, p=0.04), whereas mortality was unaffected. CONCLUSIONS: This study shows that NOAF is associated with higher rates of rehospitalization but not mortality after TAVI. Overall, patients with pre-existing AF have higher mortality.

Carbon Fixation Driven by Molecular Hydrogen Results in Chemolithoautotrophically Enhanced Growth of Helicobacter pylori.

UNLABELLED: A molecular hydrogen (H2)-stimulated, chemolithoautotrophic growth mode for the gastric pathogen Helicobacter pylori is reported. In a culture medium containing peptides and amino acids, H2-supplied cells consistently achieved 40 to 60% greater growth yield in 16 h and accumulated 3-fold more carbon from [(14)C]bicarbonate (on a per cell basis) in a 10-h period than cells without H2 Global proteomic comparisons of cells supplied with different atmospheric conditions revealed that addition of H2 led to increased amounts of hydrogenase and the biotin carboxylase subunit of acetyl coenzyme A (acetyl-CoA) carboxylase (ACC), as well as other proteins involved in various cellular functions, including amino acid metabolism, heme synthesis, or protein degradation. In agreement with this result, H2-supplied cells contained 3-fold more ACC activity than cells without H2 Other possible carbon dioxide (CO2) fixation enzymes were not up-expressed under the H2-containing atmosphere. As the gastric mucus is limited in carbon and energy sources and the bacterium lacks mucinase, this new growth mode may contribute to the persistence of the pathogen in vivo This is the first time that chemolithoautotrophic growth is described for a pathogen. IMPORTANCE: Many pathogens must survive within host areas that are poorly supplied with carbon and energy sources, and the gastric pathogen Helicobacter pylori resides almost exclusively in the nutritionally stringent mucus barrier of its host. Although this bacterium is already known to be highly adaptable to gastric niches, a new aspect of its metabolic flexibility, whereby molecular hydrogen use (energy) is coupled to carbon dioxide fixation (carbon acquisition) via a described carbon fixation enzyme, is shown here. This growth mode, which supplements heterotrophy, is termed chemolithoautotrophy and has not been previously reported for a pathogen.

A novel DNA-binding protein plays an important role in Helicobacter pylori stress tolerance and survival in the host.

The gastric pathogen Helicobacter pylori must combat chronic acid and oxidative stress. It does so via many mechanisms, including macromolecule repair and gene regulation. Mitomycin C-sensitive clones from a transposon mutagenesis library were screened. One sensitive strain contained the insertion element at the locus of hp119, a hypothetical gene. No homologous gene exists in any (non-H. pylori) organism. Nevertheless, the predicted protein has some features characteristic of histone-like proteins, and we showed that purified HP119 protein is a DNA-binding protein. A Δhp119 strain was markedly more sensitive (viability loss) to acid or to air exposure, and these phenotypes were restored to wild-type (WT) attributes upon complementation of the mutant with the wild-type version of hp119 at a separate chromosomal locus. The mutant strain was approximately 10-fold more sensitive to macrophage-mediated killing than the parent or the complemented strain. Of 12 mice inoculated with the wild type, all contained H. pylori, whereas 5 of 12 mice contained the mutant strain; the mean colonization numbers were 158-fold less for the mutant strain. A proteomic (two-dimensional PAGE with mass spectrometric analysis) comparison between the Δhp119 mutant and the WT strain under oxidative stress conditions revealed a number of important antioxidant protein differences; SodB, Tpx, TrxR, and NapA, as well as the peptidoglycan deacetylase PgdA, were significantly less expressed in the Δhp119 mutant than in the WT strain. This study identified HP119 as a putative histone-like DNA-binding protein and showed that it plays an important role in Helicobacter pylori stress tolerance and survival in the host.

Aconitase Functions as a Pleiotropic Posttranscriptional Regulator in Helicobacter pylori.

UNLABELLED: Posttranscriptional regulation in bacteria has increasingly become recognized as playing a major role in response to environmental stimuli. Aconitase is a bifunctional protein that not only acts enzymatically but also can be a posttranscriptional regulator. To investigate protein expression regulated by Helicobacter pylori AcnB in response to oxidative stress, a global proteomics study was conducted wherein the ΔacnB strain was compared to the parent strain when both strains were O2 stressed. Many proteins, including some involved in urease activity, in combating oxidative stress, and in motility, were expressed at a significantly lower level in the ΔacnB strain. A bioinformatics prediction tool was used to identify putative targets for aconitase-mediated regulation, and electrophoretic mobility shift assays demonstrated that apo-AcnB is able to bind to RNA transcripts of hpn (encoding a nickel-sequestering protein), ahpC (encoding alkyl hydroperoxide reductase), and flgR (encoding flagellum response regulator). Compared to the wild type (WT), the ΔacnB strain had decreased activities of the nickel-containing enzymes urease and hydrogenase, and this could be correlated with lower total nickel levels within ΔacnB cells. Binding of apo-AcnB to the hpn 5' untranslated region (UTR) may inhibit the expression of Hpn. In agreement with the finding that AcnB regulates the expression of antioxidant proteins such as AhpC, ΔacnB cells displayed oxidative-stress-sensitive phenotypes. The ΔacnB strain has a lesser motility ability than the WT strain, which can likely be explained by the functions of AcnB on the FlgRS-RpoN-FlgE regulatory cascade. Collectively, our results suggest a global role for aconitase as a posttranscriptional regulator in this gastric pathogen. IMPORTANCE: Bacterial survival depends on the ability of the cell to sense and respond to a variety of environmental changes. For Helicobacter pylori, responding to environmental stimuli within the gastric niche is essential for persistence and host colonization. However, there is much to be learned about the regulatory mechanisms that H. pylori employs to orchestrate its response to different stimuli. In this study, we explore the role of aconitase, a bifunctional protein that has been found to act as a posttranscriptional regulator in several other bacteria. Our results shed light on the magnitude of aconitase-mediated regulation in H. pylori, and we propose that aconitase acts as a global regulator of key genes involved in virulence.

Comparative Roles of the Two Helicobacter pylori Thioredoxins in Preventing Macromolecule Damage.

Thioredoxins are highly conserved throughout a wide range of organisms, and they are essential for the isurvival of oxygen-sensitive cells. The gastric pathogen Helicobacter pylori uses the thioredoxin system to maintain its thiol/disulfide balance. There are two thioredoxins present in H. pylori, Trx1 and Trx2 (herein referred to as TrxA and TrxC). TrxA has been shown to be important as an electron donor for some antioxidant enzymes, but the function of TrxC remains unknown (L. M. Baker, A. Raudonikiene, P. S. Hoffman, and L. B. Poole, J Bacteriol 183:1961-1973, 2001; P. Alamuri and R. J. Maier, J Bacteriol 188:5839-5850, 2006). We demonstrate that both TrxA and TrxC are important in protecting H. pylori from oxidative stress. Individual ΔtrxA and ΔtrxC deletion mutant strains each show a greater abundance of lipid peroxides and suffer more DNA damage and more protein carbonylation than the parent. Both deletion mutants were much more sensitive to O2-mediated viability loss than the parent. Unexpectedly, the oxidative DNA damage and protein carbonylation was more severe in the ΔtrxC mutant than in the ΔtrxA mutant; it had 20-fold- and 4-fold-more carbonylated protein content than the wild type and the ΔtrxA strain, respectively, after 4 h of atmospheric O2 stress. trx transcript abundance was altered by the deletion of the heterologous trx gene. The ΔtrxC mutant lacked mouse colonization ability, while the ability to colonize mouse stomachs was significantly reduced in the ΔtrxA mutant.

Host hydrogen rather than that produced by the pathogen is important for Salmonella enterica serovar Typhimurium virulence.

Salmonella enterica serovar Typhimurium utilizes molecular hydrogen as a substrate in various respiratory pathways, via H2-uptake enzymes termed Hya, Hyb, and Hyd. A different hydrogenase, the hydrogen-evolving Hyc enzyme, removes excess reductant during fermentative growth. Virulence phenotypes conferred by mutations in hyc genes, either alone or in combination with mutations in the H2-uptake enzyme genes, are addressed. Anaerobically grown ΔhycB or ΔhycC single-deletion strains were more sensitive to acid than the wild-type strain, but the Δhyc strains were like the virulent parent strain with respect to both mouse morbidity and mortality and in organ burden numbers. Even fecal-recovery numbers for both mutant strains at several time points prior to the animals succumbing to salmonellosis were like those seen with the parent. Neither hydrogen uptake nor evolution of the gas was detected in a hydrogenase quadruple-mutant strain containing deletions in the hya, hyb, hyd, and hyc genes. As previously described, a strain lacking all H2-uptake ability was severely attenuated in its virulence characteristics, and the quadruple-mutant strain had the same (greatly attenuated) phenotype. While H2 levels were greatly reduced in ceca of mice treated with antibiotics, both the ΔhycB and ΔhycC strains were still like the parent in their ability to cause typhoid salmonellosis. It seems that the level of H2 produced by the pathogen (through formate hydrogen lyase [FHL] and Hyc) is insignificant in terms of providing respiratory reductant to facilitate either organ colonization or contributions to gut growth leading to pathogenesis.