Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286–37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput.     

Crop performance, nitrogen and water use in flooded and aerobic rice

Irrigated aerobic rice is a new system being developed for lowland areas with water shortage and for favorable upland areas with access to supplementary irrigation. It entails the cultivation of nutrient-responsive cultivars in nonsaturated soil with sufficient external inputs to reach yields of 70–80% of high-input flooded rice. To obtain insights into crop performance, water use, and N use of aerobic rice, a field experiment was conducted in the dry seasons of 2002 and 2003 in the Philippines. Cultivar Apo was grown under flooded and aerobic conditions at 0 and at 150 kg fertilizer N ha^–1. The aerobic fields were flush irrigated when the soil water potential at 15-cm depth reached –30 kPa. A ¹⁵N isotope study was carried out in microplots within the 150-N plots to determine the fate of applied N. The yield under aerobic conditions with 150 kg N ha^–1 was 6.3 t ha^–1 in 2002 and 4.2 t ha^–1 in 2003, and the irrigation water input was 778 mm in 2002 and 826 mm in 2003. Compared with flooded conditions, the yield was 15 and 39% lower, and the irrigation water use 36 and 41% lower in aerobic plots in 2002 and 2003, respectively. N content at 150 kg N ha^–1 in leaves and total plant was nearly the same for aerobic and flooded conditions, indicating that crop growth under aerobic conditions was limited by water deficit and not by N deficit. Under aerobic conditions, average fertilizer N recovery was 22% in both the main field and the microplot, whereas under flooded conditions, it was 49% in the main field and 36% in the microplot. Under both flooded and aerobic conditions, the fraction of ¹⁵N that was determined in the soil after the growing season was 23%. Since nitrate contents in leachate water were negligible, we hypothesized that the N unaccounted for were gaseous losses. The N unaccounted for was higher under aerobic conditions than under flooded conditions. For aerobic rice, trials are suggested for optimizing dose and timing of N fertilizer. Also further improvements in water regime should be made to reduce crop water stress.     

Vergleichende morphologische und zytophotometrisch-autoradiographische Untersuchung der menschlichen Erythropoiese

Zusammenfassung Um die Abhängigkeit der erythropoietischen Zellformen von ihrer Stellung im Generationszyklus (G₁, S und G₂) zu untersuchen, wurde das Knochenmark von vier Normalpersonen mit der Kombination von zytophotometrischer DNS-Bestimmung (Feulgen-Photometrie) und autoradiographischer Technik mit ³H-TdR (in vitro) untersucht. Die Zellen wurden vor dieser Untersuchung in den nach Pappenheim gefärbten Ausstrichen differenziert.Sowohl in der Gruppe der basophilen Erythroblasten (E₁-E₃) als auch bei den polychromatischen Normoblasten (E₄) wurde eine postmitotische (G₁) und eine prämitotische Ruhephase (G₂) nachgewiesen. Beide Zellgruppen waren zu ca. 65% mit³H-TdR markiert (S).Unter den basophilen Erythroblasten war bei E₁ eine G₂-Phase, jedoch keine G₁-Phase nachweisbar. Demgegenüber fand sich bei E₃ eine ausgeprägte G₁-Phase, hingegen keine G₂-Phase. Bei E₂ war sowohl eine G₁-Phase als auch eine G₂-Phase erkennbar. Nach diesen Befunden stellen die Erythroblasten E₁-E₃ keine Zellgruppen mit jeweils vollständigem Generationszyklus dar, sondern sind als ein Zellkompartment zu verstehen, in dem die G₁-Phase vorwiegend durch die kleineren Zellen, die zytologisch die Merkmale der basophilen Normoblasten besitzen, und die G₂-Phase vorwiegend durch die größeren Zellen vom Typ der Proerythroblasten und Makroblasten repräsentiert wird.

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Comparative morphological and cytophotometric-autoradiographical investigation of erythropoiesis in the human

Summary In the different types of normal human nucleated red cells the stages of the cell cycle (G₁, S and G₂) were investigated by combined application of the cytophotometric determination of the DNA content (Feulgen photometry) and of autoradiographic labelling using ³H-TdR in vitro. The individual cells were identified in Pappenheim stain.In the basophilic erythroblasts (E₁-E₃) as well as in the polychromatic erythroblasts (E₄) about 62–65% of the cells were labelled (S). The unlabelled cells partly were diploid and partly were tetraploid, representing G₁ and G₂. The oxyphilic erythroblasts (E₅) mostly were diploid and unlabelled (G₁).Within the basophilic erythroblasts G₁ was demonstrated mainly in E₃, and G₂ was demonstrated mainly in E₁. In E₂, G₁ as well as G₂ were present. The results indicate that all basophilic erythroblasts belong to one cell compartment, in which G₁ is represented by the smaller cells commonly subclassified as basophilic normoblasts and G₂ is represented by the large cells usually called proerythroblasts and macroblasts.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

A flexible sigmoid function of determinate growth

A new empirical equation for the sigmoid pattern of determinate growth, 'the beta growth function', is presented. It calculates weight (w) in dependence of time, using the following three parameters: t(m), the time at which the maximum growth rate is obtained; t(e), the time at the end of growth; and w(max), the maximal value for w, which is achieved at t(e). The beta growth function was compared with four classical (logistic, Richards, Gompertz and Weibull) growth equations, and two expolinear equations. All equations described successfully the sigmoid dynamics of seed filling, plant growth and crop biomass production. However, differences were found in estimating w(max). Features of the beta function are: (1) like the Richards equation it is flexible in describing various asymmetrical sigmoid patterns (its symmetrical form is a cubic polynomial); (2) like the logistic and the Gompertz equations its parameters are numerically stable in statistical estimation; (3) like the Weibull function it predicts zero mass at time zero, but its extension to deal with various initial conditions can be easily obtained; (4) relative to the truncated expolinear equation it provides more reasonable estimates of final quantity and duration of a growth process. In addition, the new function predicts a zero growth rate at both the start and end of a precisely defined growth period. Therefore, it is unique for dealing with determinate growth, and is more suitable than other functions for embedding in process-based crop simulation models to describe the dynamics of organs as sinks to absorb assimilates. Because its parameters correspond to growth traits of interest to crop scientists, the beta growth function is suitable for characterization of environmental and genotypic influences on growth processes. However, it is not suitable for estimating maximum relative growth rate to characterize early growth that is expected to be close to exponential.     

An economic analysis of sweet sorghum cultivation for ethanol production in North China

Sweet sorghum [Sorghum bicolor (L.) Moench] is a promising non‐food energy crop. The objective of this study was to determine the economic costs and input sensitivity of sweet sorghum compared to cotton, maize, and sunflower, at two saline‐alkali sites in Shandong (Wudi County) and Inner Mongolia (Wuyuan County) provinces of China. The data were collected quantitatively based on a face‐to‐face interview with 100 and 67 sweet sorghum growers at the two sites, respectively. The sweet sorghum grown at Wudi had lower external input (5469 CNY ha^?1), higher net return (7305 CNY ha^?1) and benefit‐cost ratio (2.36) than its reference crop, cotton (18 454 CNY ha^?1; 3615 CNY ha^?1; 1.24). At Wuyuan, the sweet sorghum showed contrasting economic performance (19 541 CNY ha^?1; ?3438 CNY ha^?1; 0.91), which was largely because of the higher labor costs compared to sunflower (10 896 CNY ha^?1; 15 133 CNY ha^?1; 2.49); and maize (9108 CNY ha^?1; 14 760 CNY ha^?1; 2.76). The productivity of sweet sorghum per unit of external input costs (kg CNY^?1) was 13.12 for Wudi and only 3.26 for Wuyuan. Based on the Cobb‐Douglas production function, the external inputs of diesel and seed had a significantly positive impact on the profitability of sweet sorghum at Wudi but not at Wuyuan. However, the costs of irrigation and film cover were significantly negative. The energy crop, sweet sorghum, showed a better return to scale on investment than cotton and sunflower.     

Nitrogen economy in relay intercropping systems of wheat and cotton

Relay intercropping of wheat and cotton is practiced on a large scale in China. Winter wheat is thereby grown as a food crop from November to June and cotton as a cash crop from April to October. The crops overlap in time, growing as an intercrop, from April till June. High levels of nitrogen are applied. In this study, we analyzed the N-economy of the monocultures of cotton and wheat, and of four relay intercropping systems, differing in number of rows per strip of cotton or wheat. Field experiments were carried out from 2001/02 to 2003/04 in the Yellow River region in China. We quantified the nitrogen uptake and nitrogen use efficiency of wheat and cotton in relay intercropping systems to test if intercrops are more resource use efficient in comparison to monocrops. Nitrogen (N) yields of wheat per unit area in the four intercropping systems were lower than in the monocrop, which ranged from 203 to 288 kg ha⁻¹. The total N-uptake per unit biomass was similar between wheat in mono- and intercrops. On average, the N-yield of cotton per unit area was lower in intercrops than in monocrops, which ranged from 110 to 127 kg ha⁻¹, but the total N-uptake per unit biomass was higher in intercropped cotton, as dry matter production was reduced to a greater extent by intercropping than N-uptake. The N-uptake of cotton was diminished during the intercropping phase, but recovered partially during later growth stages. The physiological nitrogen use efficiency (IE) of wheat was not much affected by intercropping, but it was reduced in cotton, due to delayed flowering and less reproductive growth. Total N-efficiency of the system was assessed by comparing the relative nitrogen yield total (RNT), i.e. the sum of the ratio’s of total N-uptake by a component crop in the intercrop relative to the N-uptake in the monocrop, to the relative yield total. RNT ranged from 1.4 to 1.7, while the relative yield total (RYT) ranged from 1.3 to 1.4, indicating that intercrops used more nitrogen per unit production than monocrops. An analysis of the crop nitrogen balance showed that the nitrogen surplus of sole crops amounted to 220 kg ha⁻¹ for wheat and 140 kg ha⁻¹ for cotton, while in the intercropping systems, the annual N surplus exceeded 400 kg ha⁻¹. Conventional N-management in intercrops thus results in high N-surpluses that pose an environmental risk. The N management could be improved by means of a demand-based rate and timing of N applications.     

Genotype and Planting Density Effects on Rooting Traits and Yield in Cotton （Gossypium hirsutum L,）

Root density distribution of plants is a major Indicator of competition between plants and determines resource capture from the solh This experiment was conducted in 2005 at Anyang, located in the Yellow River region, Henan Province, China. Three cotton （Gossyplum hlrsutum L.） cultivars were chosen： hybrid Btcultlvar CRI46, conventional Btcultlvars CRI44 and CRI45. Six planting densities were designed, ranging from 1.5 to 12.0 plants/m^2. Root parameters such as surface area, diameter and length were analyzed by using the DT-SCAN Image analysis method. The root length density （RLD）, root average diameter and root area Index （RAI）, root surface area per unit land area, were studied. The results showed that RLD and RAI differed between genotypes; hybrid CRI46 had significantly higher （P 〈0.05） RLD and RAI values than conventlonal cultlvars, especially under low planting densities, less than 3.0 plants/m^2. The root area index （RAI） of hybrid CRI46 was 61% higher than of CRI44 and CRI45 at the flowering stage. The RLD and RAI were also significantly different （P = 0.000） between planting densities. The depth distribution of RAI showed that at Increasing planting densities RAI was Increasingly distributed in the soil layers below 50 cm. The RAI of hybrid CRI46 was for all planting densities, obviously higher than other cultivars during the flowering and boll stages. It was concluded that the hybrid had a strong advantage in root maintenance preventing premature senescence of roots. The root diameter of hybrid CRI46 had a genetically higher root diameter at planting densities lower than 6.0 plants/m^2. Good associations were found between yield and RAI In different stages. The optimum planting density ranged from 4.50 plants/m^2 to 6.75 plants/m^2 for conventional cultlvars and around 4.0-5.0 plants/m^2 for hybrids.     

Comprehensive mutation analysis in colorectal flat adenomas

Background

Flat adenomas are a subgroup of colorectal adenomas that have been associated with a distinct biology and a more aggressive clinical behavior compared to their polypoid counterparts. In the present study, we aimed to compare the mutation spectrum of 14 cancer genes, between these two phenotypes.

Methods

A consecutive series of 106 flat and 93 polypoid adenomas was analyzed retrospectively for frequently occurring mutations in “hot spot” regions of KRAS, BRAF, PIK3CA and NRAS, as well as selected mutations in CTNNB1 (β-catenin), EGFR, FBXW7 (CDC4), PTEN, STK11, MAP2K4, SMAD4, PIK3R1 and PDGFRA using a high-throughput genotyping technique. Additionally, APC was analyzed using direct sequencing.

Results

APC mutations were more frequent in polypoid adenomas compared to flat adenomas (48.5% versus 30.3%, respectively, p = 0.02). Mutations in KRAS, BRAF, NRAS, FBXW7 and CTNNB1 showed similar frequencies in both phenotypes. Between the different subtypes of flat adenomas (0-IIa, LST-F and LST-G) no differences were observed for any of the investigated genes.

Conclusion

The lower APC mutation rate in flat adenomas compared to polypoid adenomas suggests that disruption of the Wnt-pathway may occur via different mechanisms in these two phenotypes. Furthermore, in contrast to previous observations our results in this large well-defined sample set indicate that there is no significant association between the different morphological phenotypes and mutations in key genes of the RAS-RAF-MAPK pathway.     

Early Life Exposure to Famine and Colorectal Cancer Risk: A Role for Epigenetic Mechanisms

Background

Exposure to energy restriction during childhood and adolescence is associated with a lower risk of developing colorectal cancer (CRC). Epigenetic dysregulation during this critical period of growth and development may be a mechanism to explain such observations. Within the Netherlands Cohort Study on diet and cancer, we investigated the association between early life energy restriction and risk of subsequent CRC characterized by the (promoter) CpG island methylation phenotype (CIMP).

Methodology/Principal Findings

Information on diet and risk factors was collected by baseline questionnaire (n = 120,856). Three indicators of exposure were assessed: place of residence during the Hunger Winter (1944–45) and World War II years (1940–44), and father''s employment status during the Economic Depression (1932–40). Methylation specific PCR (MSP) on DNA from paraffin embedded tumor tissue was performed to determine CIMP status according to the Weisenberger markers. After 7.3 years of follow-up, 603 cases and 4631 sub-cohort members were available for analysis. Cox regression was used to calculate hazard ratios (HR) and 95% confidence intervals for CIMP+ (27.7%) and CIMP- (72.3%) tumors according to the three time periods of energy restriction, adjusted for age and gender. Individuals exposed to severe famine during the Hunger Winter had a decreased risk of developing a tumor characterized by CIMP compared to those not exposed (HR 0.65, 95%CI: 0.45–0.92). Further categorizing individuals by an index of ‘0–1’ ‘2–3’ or ‘4–7’ genes methylated in the promoter region suggested that exposure to the Hunger Winter was associated with the degree of promoter hypermethylation (‘0–1 genes methylated’ HR = 1.01, 95%CI:0.74–1.37; ‘2–3 genes methylated’ HR = 0.83, 95% CI:0.61–1.15; ‘4–7 genes methylated’ HR = 0.72, 95% CI:0.49–1.04). No associations were observed with respect to the Economic Depression and WWII years.

Conclusions

This is the first study indicating that exposure to a severe, transient environmental condition during adolescence and young adulthood may result in persistent epigenetic changes that later influence CRC development.