Investigation of an Iron-Oxidizing Microbial Mat Community Located near Aarhus, Denmark: Field Studies

We investigated the microbial community that developed at an iron seep where anoxic groundwater containing up to 250 μM Fe²⁺ flowed out of a rock wall and dense, mat-like aggregations of ferric hydroxides formed at the oxic-anoxic interface. In situ analysis with oxygen microelectrodes revealed that the oxygen concentrations in the mat were rarely more than 50% of air saturation and that the oxygen penetration depth was quite variable, ranging from <0.05 cm to several centimeters. The bulk pH of the mat ranged from 7.1 to 7.6. There appeared to be a correlation between the flow rates at different subsites of the mat and the morphotypes of the microorganisms and Fe oxides that developed. In subsites with low flow rates (<2 ml/s), the iron-encrusted sheaths of Leptothrix ochracea predominated. Miniature cores revealed that the top few millimeters of the mat consisted primarily of L. ochracea sheaths, only about 7% of which contained filaments of cells. Deeper in the mat, large particulate oxides developed, which were often heavily colonized by unicellular bacteria that were made visible by staining with acridine orange. Direct cell counts revealed that the number of bacteria increased from approximately 10⁸ to 10⁹ cells per cm³ and the total iron concentration increased from approximately 0.5 to 3 mmol/cm³ with depth in the mat. Primarily because of the growth of L. ochracea, the mat could accrete at rates of up to 3.1 mm/day at these subsites. The iron-encrusted stalks of Gallionella spp. prevailed in localized zones of the same low-flow-rate subsites, usually close to where the source water emanated from the wall. These latter zones had the lowest O₂ concentrations (<10% of the ambient concentration), confirming the microaerobic nature of Gallionella spp. In subsites with high flow rates (>6 ml/s) particulate Fe oxides were dominant; direct counts revealed that up to 10⁹ cells of primarily unicellular bacteria per cm³ were associated with these particulate oxides. These zones exhibited little vertical stratification in either the number of cells or iron concentration. Finally, mat samples incubated anaerobically in the presence of acetate or succinate exhibited significant potential for iron reduction, suggesting the possibility that a localized iron cycle could occur within the mat community.     

Activity Measurements of Planktonic Microbial and Microfouling Communities in a Eutrophic Estuary

[³H]thymidine incorporation, the rate of reduction of iodonitrotetrazolium violet (INT) to INT formazan normalized to DNA, and the ratio of ATP to DNA were adapted to measure the activity of attached and unattached microbial assemblages of Bayboro Harbor, Fla. Activity measurements by [³H]thymidine incorporation were made of cells attached to polystyrene culture dishes, in unfiltered water samples, and in the <1-μm-filtered fraction. In most cases, the activity of attached cells was greater than that of unattached cells either in unfiltered water samples or in the <1-μm fraction. The calculated thymidine incorporation rates for cells in the >1-μm fraction were higher than those for cells either in unfiltered water or in the <1-μm-filtered fraction. By the rate of reduction of INT to INT formazan normalized to DNA and by ATP-to-DNA ratios, attached cells were also more active than cells in unfiltered water samples. These results indicate that the microenvironment afforded by attachment is a more beneficial habitat for microbial growth. Reasons for greater activity by natural populations of attached bacteria are discussed.     

Purification and structural characterization of a flavoprotein induced by iron limitation in Methanobacterium thermoautotrophicum Marburg.

Cells of Methanobacterium thermoautotrophicum (strain Marburg) grown under iron-limiting conditions were found to synthesize a soluble polypeptide as one of the major cell proteins. This polypeptide purified as a homotetramer (170 kDa [subunit molecular mass, 43 kDa]) had a UV-visible spectrum typical of flavoproteins and contained 0.7 mol of flavin mononucleotide per mol of monomer. Quantitative analysis by immunoblotting with polyclonal antibodies indicated that the flavoprotein, which amounts to about 0.6% of soluble cell protein under iron-sufficient conditions (> or = 50 microM Fe2+), was induced fivefold by iron limitation (< 12 microM Fe2+). The flavoprotein-encoding gene, fprA, was cloned and sequenced. Sequence analysis revealed a well-conserved archaebacterial consensus promoter upstream of fprA, a flavodoxin signature within fprA, and 28% amino acid identity with a putative flavin mononucleotide-containing protein of Rhodobacter capsulatus which is found within an operon involved in nitrogen fixation. A possible physiological function for the flavoprotein is discussed.     

Electron Spin Resonance in ATP and RNA

Cu⁺⁺, Mn⁺⁺, and Fe⁺⁺⁺ account for the electron spin resonances observed in certain samples of ATP and RNA. The copper ion seems more loosely bound to these substances than either iron or manganese. A striking similarity is observed between the manganese spectra in manganese RNA, ATP, and ADP suggesting that the binding sites are similar in the three compounds. The similarity of the e.s.r. spectra of iron ATP and of iron and manganese RNA, except for hyperfine spectrum (hfs) in the latter, suggests that the two ions bind similarly in the two compounds. A detailed interpretation of the spectra is lacking however and these conclusions can only be tentative. When manganese TPP and ATP are heated or pH changed the e.s.r. alters indicating a change in the environment of the ion. The sharp 6 line manganese spectrum in both TPP and ATP at pH 1 suggests an almost “free” ion at this pH in the sense of an almost isotropic average environment.     

Training elite youth soccer players: area per player in small-sided games to replicate the match demands

The aim was to determine the area per player (ApP, m² × player) in small- or large-sided games to replicate the official match demands in elite youth soccer players. Two hundred and twenty-eight players (U15 = 36, U16 = 48, U17 = 49, U18 = 37 and U19 = 58) were monitored during both training (12 183 individual samples) and matches (683 individual samples) across five seasons. Relative (m × min^-1) total (TD), high-speed running (HSR), very high-speed running (VHSR), sprint and acceleration/deceleration (Acc/Dec) distance were collected. Between-category and between-position comparisons were performed. Area per player was moderately correlated (P < 0.05) with TD (r = 0.401), large (r = 0.621) with HSR, and very largely with VHSR (r = 0.744) and sprint (r = 0.723). An inverse small (r = -0.232; P = 0.039) correlation for Acc/Dec was found. The area per player to replicate the match demands was 158 ± 18, 182 ± 32, 197 ± 37, 212 ± 42 and 156 ± 25 m² × player for TD, HSR, VHSR, sprint and Acc/Dec, respectively. Moderate to very large (ES: 0.79 to 4.66) differences in the area per player across metrics were observed, with sprint > VHSR > HSR > TD = Acc/Dec. Trivial to very large (ES: 0.01 to 2.67) between-category differences in area per player across the same metric were found, with U15 and U16 requiring a larger area per player than other age categories. These findings may help practitioners to recreate the desired external load outcomes with regards to positional match-play demands using specific area per player in small- or large-sided games in youth elite soccer players from U15 to U19.     

Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N₂ grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered.     

Iron stores at birth in a full-term normal birth weight birth cohort with a low level of inflammation

Iron stores at birth are essential to meet iron needs during the first 4–6 months of life. The present study aimed to investigate iron stores in normal birth weight, healthy, term neonates. Umbilical cord blood samples were collected from apparently normal singleton vaginal deliveries (n=854). Subjects were screened and excluded if C-reactive protein (CRP) > 5 mg/l or α1-acid glycoprotein (AGP) > 1 g/l, preterm (<37 complete weeks), term < 2500g or term > 4000g. In total, 762 samples were included in the study. Serum ferritin, soluble transferrin receptor (sTfR), hepcidin, and erythropoietin (EPO) were measured in umbilical cord blood samples; total body iron (TBI) (mg/kg) was calculated using sTfR and ferritin concentrations. A total of 19.8% newborns were iron deficient (ferritin 35 μg/l) and an additional 46.6% had insufficient iron stores (ferritin < 76 μg/l). There was a positive association between serum ferritin and sTfR, hepcidin, and EPO. Gestational age was positively associated with ferritin, sTfR, EPO, and hepcidin. In conclusion, we demonstrate a high prevalence of insufficient iron stores in a Chinese birth cohort. The value of cord sTfR and TBI in the assessment of iron status in the newborn is questionable, and reference ranges need to be established.     

Evaluation of Two Highly-Multiplexed Custom Panels for Massively Parallel Semiconductor Sequencing on Paraffin DNA

Background—Aim

Massively parallel sequencing (MPS) holds promise for expanding cancer translational research and diagnostics. As yet, it has been applied on paraffin DNA (FFPE) with commercially available highly multiplexed gene panels (100s of DNA targets), while custom panels of low multiplexing are used for re-sequencing. Here, we evaluated the performance of two highly multiplexed custom panels on FFPE DNA.

Methods

Two custom multiplex amplification panels (B, 373 amplicons; T, 286 amplicons) were coupled with semiconductor sequencing on DNA samples from FFPE breast tumors and matched peripheral blood samples (n samples: 316; n libraries: 332). The two panels shared 37% DNA targets (common or shifted amplicons). Panel performance was evaluated in paired sample groups and quartets of libraries, where possible.

Results

Amplicon read ratios yielded similar patterns per gene with the same panel in FFPE and blood samples; however, performance of common amplicons differed between panels (p<0.001). FFPE genotypes were compared for 1267 coding and non-coding variant replicates, 999 out of which (78.8%) were concordant in different paired sample combinations. Variant frequency was highly reproducible (Spearman’s rho 0.959). Repeatedly discordant variants were of high coverage / low frequency (p<0.001). Genotype concordance was (a) high, for intra-run duplicates with the same panel (mean±SD: 97.2±4.7, 95%CI: 94.8–99.7, p<0.001); (b) modest, when the same DNA was analyzed with different panels (mean±SD: 81.1±20.3, 95%CI: 66.1–95.1, p = 0.004); and (c) low, when different DNA samples from the same tumor were compared with the same panel (mean±SD: 59.9±24.0; 95%CI: 43.3–76.5; p = 0.282). Low coverage / low frequency variants were validated with Sanger sequencing even in samples with unfavourable DNA quality.

Conclusions

Custom MPS may yield novel information on genomic alterations, provided that data evaluation is adjusted to tumor tissue FFPE DNA. To this scope, eligibility of all amplicons along with variant coverage and frequency need to be assessed.     

Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants

The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log₁₀ copies per million PBMC, p<0.001; ET: -1.04±0.11 log₁₀ DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log₁₀ DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log₁₀ DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible.     

Dephytinisation with Intrinsic Wheat Phytase and Iron Fortification Significantly Increase Iron Absorption from Fonio (Digitaria exilis) Meals in West African Women

Low iron and high phytic acid content make fonio based meals a poor source of bioavailable iron. Phytic acid degradation in fonio porridge using whole grain cereals as phytase source and effect on iron bioavailability when added to iron fortified fonio meals were investigated. Grains, nuts and seeds collected in Mali markets were screened for phytic acid and phytase activity. We performed an iron absorption study in Beninese women (n = 16), using non-dephytinised fonio porridge (FFP) and dephytinised fonio porridge (FWFP; 75% fonio-25% wheat), each fortified with ⁵⁷Fe or ⁵⁸Fe labeled FeSO₄. Iron absorption was quantified by measuring the erythrocyte incorporation of stable iron isotopes. Phytic acid varied from 0.39 (bambara nut) to 4.26 g/100 g DM (pumpkin seed), with oilseeds values higher than grains and nuts. Phytase activity ranged from 0.17±1.61 (fonio) to 2.9±1.3 phytase unit (PU) per g (whole wheat). Phytic acid was almost completely degraded in FWFP after 60 min of incubation (pH≈5.0, 50°C). Phytate∶iron molar ratios decreased from 23.7∶1 in FFP to 2.7∶1 in FWFP. Iron fortification further reduced phytate∶iron molar ratio to 1.9∶1 in FFP and 0.3∶1 in FWFP, respectively. Geometric mean (95% CI) iron absorption significantly increased from 2.6% (0.8–7.8) in FFP to 8.3% (3.8–17.9) in FWFP (P<0.0001). Dephytinisation of fonio porridge with intrinsic wheat phytase increased fractional iron absorption 3.2 times, suggesting it could be a possible strategy to decrease PA in cereal-based porridges.     

Fast and Inexpensive Detection of Total and Extractable Element Concentrations in Aquatic Sediments Using Near-Infrared Reflectance Spectroscopy (NIRS)

Adequate biogeochemical characterization and monitoring of aquatic ecosystems, both for scientific purposes and for water management, pose high demands on spatial and temporal replication of chemical analyses. Near-infrared reflectance spectroscopy (NIRS) may offer a rapid, low-cost and reproducible alternative to standard analytical sample processing (digestion or extraction) and measuring techniques used for the chemical characterization of aquatic sediments. We analyzed a total of 191 sediment samples for total and NaCl-extractable concentrations of Al, Ca, Fe, K, Mg, Mn, N, Na, P, S, Si, and Zn as well as oxalate- extractable concentrations of Al, Fe, Mn and P. Based on the NIR spectral data and the reference values, calibration models for the prediction of element concentrations in unknown samples were developed and tested with an external validation procedure. Except Mn, all prediction models of total element concentrations were found to be acceptable to excellent (ratio of performance deviation: RPD 1.8–3.1). For extractable element fractions, viable model precision could be achieved for NaCl-extractable Ca, K, Mg, NH₄ ⁺-N, S and Si (RPD 1.7–2.2) and oxalate-extractable Al, Fe and P (RPD 1.9–2.3). For those elements that showed maximum total values below 3 g kg⁻¹ prediction models were found to become increasingly critical (RPD <2.0). Low concentrations also limited the performance of NIRS calibrations for extracted elements, with critical concentration thresholds <0.1 g kg⁻¹ and 3.3 g kg⁻¹ for NaCl and oxalate extractions, respectively. Thus, reliable NIRS measurements of trace metals are restricted to sediments with high metal content. Nevertheless, we demonstrated the suitability of NIRS measurements to determine a large array of chemical properties of aquatic sediments. The results indicate great potential of this fast technique as an analytical tool to better understand the large spatial and temporal variation of sediment characteristics in an economically viable way.     

Microbiological Survey of Adirondack Lakes with Various pH Values

Nine high-altitude oligotrophic Adirondack lakes in upstate New York having water of pH 4.3 to 7.0 were surveyed for total bacterial numbers and possible adaptation of the microbial communities to environmental pH. The number of heterotrophic bacteria from water samples recoverable on standard plate count agar were low (10¹ to 10³ per ml) for most of the lakes. Acridine orange direct counts were approximately two orders of magnitude higher than plate counts for each lake. Sediment aerobic heterotrophs recovered on standard plate count agar ranged from 1.4 × 10⁴ to 1.3 × 10⁶ per g of sediment. Direct epifluorescence counts of bacteria in sediment samples ranged from 3.0 × 10⁶ to 1.4 × 10⁷ per g. Low density values were consistent with the oligotrophic nature of all the lakes surveyed. There were no apparent differences in numbers of bacteria originally isolated at pH 5.0 and pH 7.0 between circumneutral lakes (pH > 6.0) and acidic lakes (pH < 5.0). Approximately 1,200 isolates were recultured over a range of pH from 3.0 to 7.0. Regardless of the original isolation pH (pH 5.0 or pH 7.0), less than 10% of the isolates grew at pH < 5.0. Those originally isolated at pH 5.0 also grew at pH 6.0 and 7.0. Those originally isolated at pH 7.0 preferred pH 7.0, with 98% able to grow at pH 6.0 and 44% able to grow at pH 5.0. A chi-square contingency test clearly showed (P < 0.005) that two distinct heterotrophic populations had been originally isolated at pH 5.0 and pH 7.0, although there is undoubtedly some overlap between the two populations.     

Twisting DNA by salt

The structure and properties of DNA depend on the environment, in particular the ion atmosphere. Here, we investigate how DNA twist -one of the central properties of DNA- changes with concentration and identity of the surrounding ions. To resolve how cations influence the twist, we combine single-molecule magnetic tweezer experiments and extensive all-atom molecular dynamics simulations. Two interconnected trends are observed for monovalent alkali and divalent alkaline earth cations. First, DNA twist increases monotonously with increasing concentration for all ions investigated. Second, for a given salt concentration, DNA twist strongly depends on cation identity. At 100 mM concentration, DNA twist increases as Na⁺ < K⁺ < Rb⁺ < Ba²⁺ < Li⁺ ≈ Cs⁺ < Sr²⁺ < Mg²⁺ < Ca²⁺. Our molecular dynamics simulations reveal that preferential binding of the cations to the DNA backbone or the nucleobases has opposing effects on DNA twist and provides the microscopic explanation of the observed ion specificity. However, the simulations also reveal shortcomings of existing force field parameters for Cs⁺ and Sr²⁺. The comprehensive view gained from our combined approach provides a foundation for understanding and predicting cation-induced structural changes both in nature and in DNA nanotechnology.     

Effects of particulate matter exposure on blood 5-hydroxymethylation: results from the Beijing truck driver air pollution study

Previous studies have reported epigenetic changes induced by environmental exposures. However, previous investigations did not distinguish 5-methylcytosine (5mC) from a similar oxidative form with opposite functions, 5-hydroxymethylcytosine (5hmC). Here, we measured blood DNA global 5mC and 5hmC by ELISA and used adjusted mixed-effects regression models to evaluate the effects of ambient PM₁₀ and personal PM_2.5 and its elemental components—black carbon (BC), aluminum (Al), calcium (Ca), potassium (K), iron (Fe), sulfur (S), silicon (Si), titanium (Ti), and zinc (Zn)—on blood global 5mC and 5hmC levels. The study was conducted in 60 truck drivers and 60 office workers in Beijing, China from The Beijing Truck Driver Air Pollution Study at 2 exams separated by one to 2 weeks. Blood 5hmC level (0.08%) was ∼83-fold lower than 5mC (6.61%). An inter-quartile range (IQR) increase in same-day PM₁₀ was associated with increases in 5hmC of 26.1% in office workers (P = 0.004), 20.2% in truck drivers (P = 0.014), and 21.9% in all participants combined (P < 0.001). PM₁₀ effects on 5hmC were increasingly stronger when averaged over 4, 7, and 14 d preceding assessment (up to 132.6% for the 14-d average in all participants, P < 0.001). PM₁₀ effects were also significant after controlling for multiple testing (family-wise error rate; FWER < 0.05). 5hmC was not correlated with personal measures of PM_2.5 and elemental components (FWER > 0.05). 5mC showed no correlations with PM₁₀, PM_2.5, and elemental components measures (FWER > 0.05). Our study suggests that exposure to ambient PM₁₀ affects 5hmC over time, but not 5mC. This finding demonstrates the need to differentiate 5hmC and 5mC in environmental studies of DNA methylation.     

The Relation of Hepcidin to Iron Disorders,Inflammation and Hemoglobin in Chronic Kidney Disease

The metabolism of hepcidin is profoundly modified in chronic kidney disease (CKD). We investigated its relation to iron disorders, inflammation and hemoglobin (Hb) level in 199 non-dialyzed, non-transplanted patients with CKD stages 1–5. All had their glomerular filtration rate measured by ⁵¹Cr-EDTA renal clearance (mGFR), as well as measurements of iron markers including hepcidin and of erythropoietin (EPO). Hepcidin varied from 0.2 to 193 ng/mL. The median increased from 23.3 ng/mL [8.8–28.7] to 36.1 ng/mL [14.1–92.3] when mGFR decreased from ≥60 to <15 mL/min/1.73 m² (p = 0.02). Patients with absolute iron deficiency (transferrin saturation (TSAT) <20% and ferritin <40 ng/mL) had the lowest hepcidin levels (5.0 ng/mL [0.7–11.7]), and those with a normal iron profile (TSAT ≥20% and ferritin ≥40), the highest (34.5 ng/mL [23.7–51.6]). In multivariate analysis, absolute iron deficiency was associated with lower hepcidin values, and inflammation combined with a normal or functional iron profile with higher values, independent of other determinants of hepcidin concentration, including EPO, mGFR, and albuminemia. The hepcidin level, although it rose overall when mGFR declined, collapsed in patients with absolute iron deficiency. There was a significant interaction with iron status in the association between Hb and hepcidin. Except in absolute iron deficiency, hepcidin’s negative association with Hb level indicates that it is not down-regulated in CKD anemia.     

High Abundances of Potentially Active Ammonia-Oxidizing Bacteria and Archaea in Oligotrophic,High-Altitude Lakes of the Sierra Nevada,California, USA

Nitrification plays a central role in the nitrogen cycle by determining the oxidation state of nitrogen and its subsequent bioavailability and cycling. However, relatively little is known about the underlying ecology of the microbial communities that carry out nitrification in freshwater ecosystems—and particularly within high-altitude oligotrophic lakes, where nitrogen is frequently a limiting nutrient. We quantified ammonia-oxidizing archaea (AOA) and bacteria (AOB) in 9 high-altitude lakes (2289–3160 m) in the Sierra Nevada, California, USA, in relation to spatial and biogeochemical data. Based on their ammonia monooxygenase (amoA) genes, AOB and AOA were frequently detected. AOB were present in 88% of samples and were more abundant than AOA in all samples. Both groups showed >100 fold variation in abundance between different lakes, and were also variable through time within individual lakes. Nutrient concentrations (ammonium, nitrite, nitrate, and phosphate) were generally low but also varied across and within lakes, suggestive of active internal nutrient cycling; AOB abundance was significantly correlated with phosphate (r² = 0.32, p<0.1), whereas AOA abundance was inversely correlated with lake elevation (r² = 0.43, p<0.05). We also measured low rates of ammonia oxidation—indicating that AOB, AOA, or both, may be biogeochemically active in these oligotrophic ecosystems. Our data indicate that dynamic populations of AOB and AOA are found in oligotrophic, high-altitude, freshwater lakes.     

Metal Reduction and Iron Biomineralization by a Psychrotolerant Fe(III)-Reducing Bacterium, Shewanella sp. Strain PV-4

A marine psychrotolerant, dissimilatory Fe(III)-reducing bacterium, Shewanella sp. strain PV-4, from the microbial mat at a hydrothermal vent of Loihi Seamount in the Pacific Ocean has been further characterized, with emphases on metal reduction and iron biomineralization. The strain is able to reduce metals such as Fe(III), Co(III), Cr(VI), Mn(IV), and U(VI) as electron acceptors while using lactate, formate, pyruvate, or hydrogen as an electron donor. Growth during iron reduction occurred over the pH range of 7.0 to 8.9, a sodium chloride range of 0.05 to 5%, and a temperature range of 0 to 37°C, with an optimum growth temperature of 18°C. Unlike mesophilic dissimilatory Fe(III)-reducing bacteria, which produce mostly superparamagnetic magnetite (<35 nm), this psychrotolerant bacterium produces well-formed single-domain magnetite (>35 nm) at temperatures from 18 to 37°C. The genome size of this strain is about 4.5 Mb. Strain PV-4 is sensitive to a variety of commonly used antibiotics except ampicillin and can acquire exogenous DNA (plasmid pCM157) through conjugation.     

Genome-Wide Investigation of DNA Methylation Marks Associated with FV Leiden Mutation

In order to investigate whether DNA methylation marks could contribute to the incomplete penetrance of the FV Leiden mutation, a major genetic risk factor for venous thrombosis (VT), we measured genome-wide DNA methylation levels in peripheral blood samples of 98 VT patients carrying the mutation and 251 VT patients without the mutation using the dedicated Illumina HumanMethylation450 array. The genome-wide analysis of 388,120 CpG probes identified three sites mapping to the SLC19A2 locus whose DNA methylation levels differed significantly (p<3 10⁻⁸) between carriers and non-carriers. The three sites replicated (p<2 10⁻⁷) in an independent sample of 214 individuals from five large families ascertained on VT and FV Leiden mutation among which 53 were carriers and 161 were non-carriers of the mutation. In both studies, these three CpG sites were also associated (2.33 10⁻¹¹<p<3.02 10⁻⁴) with biomarkers of the Protein C pathway known to be influenced by the FV Leiden mutation. A comprehensive linkage disequilibrium (LD) analysis of the whole locus revealed that the original associations were due to LD between the FV Leiden mutation and a block of single nucleotide polymorphisms (SNP) located in SLC19A2. After adjusting for this block of SNPs, the FV Leiden mutation was no longer associated with any CpG site (p>0.05). In conclusion, our work clearly illustrates some promises and pitfalls of DNA methylation investigations on peripheral blood DNA in large epidemiological cohorts. DNA methylation levels at SLC19A2 are influenced by SNPs in LD with FV Leiden, but these DNA methylation marks do not explain the incomplete penetrance of the FV Leiden mutation.     

Non-Heme Iron Absorption and Utilization from Typical Whole Chinese Diets in Young Chinese Urban Men Measured by a Double-Labeled Stable Isotope Technique

BackgroundThis study was to observe the non-heme iron absorption and biological utilization from typical whole Chinese diets in young Chinese healthy urban men, and to observe if the iron absorption and utilization could be affected by the staple food patterns of Southern and Northern China.ResultsNon-heme iron intake values overall, and in the rice and steamed buns groups were 12.8 ±2.1, 11.3±1.3 and 14.3±1.5 mg, respectively; the mean ⁵⁷Fe absorption rates were 11±7%, 13±7%, and 8±4%, respectively; and the mean infused ⁵⁸Fe utilization rates were 85±8%, 84±6%, and 85±10%, respectively. There was no significantly difference in the iron intakes, and ⁵⁷Fe absorption and infused ⁵⁸Fe utilization rates between rice and steamed buns groups (all P>0.05).ConclusionWe present the non-heme iron absorption and utilization rates from typical whole Chinese diets among young Chinese healthy urban men, which was not affected by the representative staple food patterns of Southern and Northern China. This study will provide a basis for the setting of Chinese iron DRIs.     

Assay and characteristics of the iron binding moiety of reticulocyte endocytic vesicles

Summary A⁵⁹Fe assay was designed to detect an Fe(III) binding capacity in NP-40 solubilized proteins from rabbit reticulocyte endocytic vesicles. The iron binding capacity had an apparent molecular weight as determined by gel exclusion chromatography of 450,000 daltons. The iron binding moiety coincided with the major nontransferrin iron-containing material of endocytic vesicles labeled in vivo by incubation of cells with⁵⁹Fe,¹²⁵I-labeled transferrin. The material solubilized from vesicles with NP-40 exhibited two classes of saturable binding sites, one with an association constant for⁵⁹Fe-citrate of 3.63×10⁹ m ^–1 and with 6.6×10^–12 moles of iron bound per mg protein and the other with a constant of 3.96×10⁸ m ^–1 and 1.0×10^–12 moles of iron bound per mg protein. These affinities are sufficient to satisfy the sobulility characteristics of Fe(III) at pH 5.0. Most of the⁵⁹Fe bound both in vivo and in vitro to the iron binding moiety could be displaced with⁵⁶Fe and an equivalent amount of⁵⁹Fe could subsequently be rebound in vitro. The iron binding assay was adopted to vesicle proteins separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to nitrocellulose and revealed an iron binding activity of molecular weight approximately 95,000 daltons.