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That silicon is an important element in global biogeochemical cycles is widely recognised. Recently, its relevance for global crop production has gained increasing attention in light of possible deficits in plant-available Si in soil. Silicon is beneficial for plant growth and is taken up in considerable amounts by crops like rice or wheat. However, plants differ in the way they take up silicic acid from soil solution, with some species rejecting silicic acid while others actively incorporate it. Yet because the processes governing Si uptake and regulation are not fully understood, these classifications are subject to intense debate. To gain a new perspective on the processes involved, we investigated the dependence of silicon stable isotope fractionation on silicon uptake strategy, transpiration, water use, and Si transfer efficiency. Crop plants with rejective (tomato, Solanum lycopersicum, and mustard, Sinapis alba) and active (spring wheat, Triticum aestivum) Si uptake were hydroponically grown for 6 weeks. Using inductively coupled plasma mass spectrometry, the silicon concentration and isotopic composition of the nutrient solution, the roots, and the shoots were determined. We found that measured Si uptake does not correlate with the amount of transpired water and is thus distinct from Si incorporation expected for unspecific passive uptake. We interpret this lack of correlation to indicate a highly selective Si uptake mechanism. All three species preferentially incorporated light 28Si, with a fractionation factor 1000×ln (α) of −0.33 ‰ (tomato), −0.55 ‰ (mustard), and −0.43 ‰ (wheat) between growth medium and bulk plant. Thus, even though the rates of active and passive Si root uptake differ, the physico-chemical processes governing Si uptake and stable isotope fractionation do not. We suggest that isotope fractionation during root uptake is governed by a diffusion process. In contrast, the transport of silicic acid from the roots to the shoots depends on the amount of silicon previously precipitated in the roots and the presence of active transporters in the root endodermis, facilitating Si transport into the shoots. Plants with significant biogenic silica precipitation in roots (mustard and wheat) preferentially transport silicon depleted in 28Si into their shoots. If biogenic silica is not precipitated in the roots, Si transport is dominated by a diffusion process, and hence light silicon 28Si is preferentially transported into the tomato shoots. This stable Si isotope fingerprinting of the processes that transfer biogenic silica between the roots and shoots has the potential to track Si availability and recycling in soils and to provide a monitor for efficient use of plant-available Si in agricultural production.

Colonization of Azotobacter chroococcum Mac 27 strain on wheat roots was monitored using double antibody sandwich enzyme linked immunosorbent assay (DAS ELISA) and immuno-fluorescence in terms of colonization sites, migration and survival of the bacteria. Furthermore, the effectivity of inoculation of A. chroococcum and Pantoea agglomerans D5/23 strain on wheat plant parameters under greenhouse conditions was investigated. Studies with DAS ELISA revealed that root tip had significant titre of inoculants as compared to the basal root parts. A. chroococcum colonized roots as well as soil and also migrated along with roots. An overall increase in plant growth was observed with inoculation of these bacteria. Both A. chroococcum and P. agglomerans were found to increase plant dry matter, nitrogen and phosphate uptake.

Phosphorus (P) availability severely limits plant growth due to its immobility and inaccessibility in soils. Yet, visualization and measurements of P uptake from different root types or regions in soil are methodologically challenging. Here, we explored the potential of phosphor imaging combined with local injection of radioactive 33 P to quantitatively visualize P uptake and translocation along roots of maize grown in soils. Rhizoboxes (20 × 40 × 1 cm) were filled with sandy field soil or quartz sand, with one maize plant per box. Soil compartments were created using a gravel layer to restrict P transfer. After 2 weeks, a compartment with the tip region of a seminal root was labeled with a NaH 2 33 PO4 solution containing 12 MBq of 33 P. Phosphor imaging captured root P distribution at 45 min, 90 min, 135 min, 180 min, and 24 h post-labeling. After harvest, 33 P levels in roots and shoots were quantified. 33 P uptake exhibited a 50% increase in quartz sand compared to sandy soil, likely attributed to higher P adsorption to the sandy soil matrix than to quartz sand. Notably, only 60% of the absorbed 33 P was translocated to the shoot, with the remaining 40% directed to growing root tips of lateral or seminal roots. Phosphor imaging unveiled a continuous rise in 33 P signal in the labeled seminal root from immediate post-labeling until 24 h after labeling. The highest 33 P activities were concentrated just above the labeled compartment, diminishing in locations farther away. Emerging laterals from the labeled root served as strong sinks for 33 P, while a portion was also transported to other seminal roots. Our study quantitatively visualized 33 P uptake and translocation dynamics, facilitating future investigations into diverse root regions/types and varying plant growth conditions. This improves our understanding of the significance of different P sources for plant nutrition and potentially enhances models of plant P uptake., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Holz, Mundschenk, Pusch, Remus, Dubbert, Oburger, Staudinger, Wissuwa and Zarebanadkouki.)

Simulations of human-technology interaction in the context of product development require comprehensive knowledge of biomechanical in vivo behavior. To obtain this knowledge for the abdomen, we measured the continuous mechanical responses of the abdominal soft tissue of ten healthy participants in different lying positions anteriorly, laterally, and posteriorly under local compression depths of up to 30 mm. An experimental setup consisting of a mechatronic indenter with hemispherical tip and two time-of-flight (ToF) sensors for optical 3D displacement measurement of the surface was developed for this purpose. To account for the impact of muscle tone, experiments were conducted with both controlled activation and relaxation of the trunk muscles. Surface electromyography (sEMG) was used to monitor muscle activation levels. The obtained data sets comprise the continuous force-displacement data of six abdominal measurement regions, each synchronized with the local surface displacements resulting from the macro-indentation, and the bipolar sEMG signals at three key trunk muscles. We used inverse finite element analysis (FEA), to derive sets of nonlinear material parameters that numerically approximate the experimentally determined soft tissue behaviors. The physiological standard values obtained for all participants after data processing served as reference data. The mean stiffness of the abdomen was significantly different when the trunk muscles were activated or relaxed. No significant differences were found between the anterior-lateral measurement regions, with exception of those centered on the linea alba and centered on the muscle belly of the rectus abdominis below the intertubercular plane. The shapes and areas of deformation of the skin depended on the region and muscle activity. Using the hyperelastic Ogden model, we identified unique material parameter sets for all regions. Our findings confirmed that, in addition to the indenter force-displacement data, knowledge about tissue deformation is necessary to reliably determine unique material parameter sets using inverse FEA. The presented results can be used for finite element (FE) models of the abdomen, for example, in the context of orthopedic or biomedical product developments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Remus, Sure, Selkmann, Uttich and Bender.)

Aims: High Phosphorus (P) efficiencies such as internal P utilization efficiency (PUE) and P acquisition efficiency (PAE) are crucial for upland rice production, particularly on highly P-fixing soils like Andosols. While the effect of root traits associated with high PAE in upland rice has been studied intensively, less attention has been given to the origin of P (native soil-P versus fertilizer-P) taken up by plants when evaluating differences in P efficiency. Here we aim to evaluate the efficiency of different upland rice genotypes to acquire native soil-P and fertilizer-P., Methods: Four upland rice genotypes with varying PAE were grown in an Andosol at low- and high-P fertilization level and harvested 9 and 34 days after emergence. Fertilizer-P was labeled with 33 P to distinguish between the efficiency to acquire P originating from native soil and fertilizer by measuring plant P uptake., Results: Increased fertilizer supply enhanced native soil-P uptake. Under low-P conditions the genotype DJ123 showed a superior PAE and an increased acquisition of native soil-P while AB199 was identified to have a superior internal PUE under P deficient conditions. Differences between genotypes in overall PAE under high-P conditions were not significant but the distinction of P sources showed that genotype DJ123 acquired significantly more native soil-P per unit root than all other genotypes., Conclusions: Our results indicate that variations in PAE among genotypes are associated with their ability to access native soil-P. DJ123 emerged as the most adept genotype in acquiring sparingly soluble native soil-P and future studies should unravel the rhizosphere processes underlying increased PAE of native soil-P., Supplementary Information: The online version contains supplementary material available at 10.1007/s42729-024-01774-1., Competing Interests: Conflict of InterestsOn behalf of all authors, the corresponding author states that there is no conflict of interest., (© The Author(s) 2024.)

Most spine models belong to either the musculoskeletal multibody (MB) or finite element (FE) method. Recently, coupling of MB and FE models has increasingly been used to combine advantages of both methods. Active hybrid FE-MB models, still rarely used in spine research, avoid the interface and convergence problems associated with model coupling. They provide the inherent ability to account for the full interplay of passive and active mechanisms for spinal stability. In this paper, we developed and validated a novel muscle-driven forward dynamic active hybrid FE-MB model of the lumbosacral spine (LSS) in ArtiSynth to simultaneously calculate muscle activation patterns, vertebral movements, and internal mechanical loads. The model consisted of the rigid vertebrae L1-S1 interconnected with hyperelastic fiber-reinforced FE intervertebral discs, ligaments, facet joints, and force actuators representing the muscles. Morphological muscle data were implemented via a semi-automated registration procedure. Four auxiliary bodies were utilized to describe non-linear muscle paths by wrapping and attaching the anterior abdominal muscles. This included an abdominal plate whose kinematics was optimized using motion capture data from upper body movements. Intra-abdominal pressure was calculated from the forces of the abdominal muscles compressing the abdominal cavity. For the muscle-driven approach, forward dynamics assisted data tracking was used to predict muscle activation patterns that generate spinal postures and balance the spine without prescribing accurate spinal kinematics. During calibration, the maximum specific muscle tension and spinal rhythms resulting from the model dynamics were evaluated. To validate the model, load cases were simulated from -10° extension to +30° flexion with weights up to 20 kg in both hands. The biomechanical model responses were compared with in vivo literature data of intradiscal pressures, intra-abdominal pressures, and muscle activities. The results demonstrated high agreement with this data and highlight the advantages of active hybrid modeling for the LSS. Overall, this new self-contained tool provides a robust and efficient estimation of LSS biomechanical responses under in vivo similar loads, for example, to improve pain treatment by spinal stabilization therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Remus, Selkmann, Lipphaus, Neumann and Bender.)

Background: Managed grasslands are global sources of atmospheric methanol, which is one of the most abundant volatile organic compounds in the atmosphere and promotes oxidative capacity for tropospheric and stratospheric ozone depletion. The phyllosphere is a favoured habitat of plant-colonizing methanol-utilizing bacteria. These bacteria also occur in the rhizosphere, but their relevance for methanol consumption and ecosystem fluxes is unclear. Methanol utilizers of the plant-associated microbiota are key for the mitigation of methanol emission through consumption. However, information about grassland plant microbiota members, their biodiversity and metabolic traits, and thus key actors in the global methanol budget is largely lacking., Results: We investigated the methanol utilization and consumption potentials of two common plant species (Festuca arundinacea and Taraxacum officinale) in a temperate grassland. The selected grassland exhibited methanol formation. The detection of 13 C derived from 13 C-methanol in 16S rRNA of the plant microbiota by stable isotope probing (SIP) revealed distinct methanol utilizer communities in the phyllosphere, roots and rhizosphere but not between plant host species. The phyllosphere was colonized by members of Gamma- and Betaproteobacteria. In the rhizosphere, 13 C-labelled Bacteria were affiliated with Deltaproteobacteria, Gemmatimonadates, and Verrucomicrobiae. Less-abundant 13 C-labelled Bacteria were affiliated with well-known methylotrophs of Alpha-, Gamma-, and Betaproteobacteria. Additional metagenome analyses of both plants were consistent with the SIP results and revealed Bacteria with methanol dehydrogenases (e.g., MxaF1 and XoxF1-5) of known but also unusual genera (i.e., Methylomirabilis, Methylooceanibacter, Gemmatimonas, Verminephrobacter). 14 C-methanol tracing of alive plant material revealed divergent potential methanol consumption rates in both plant species but similarly high rates in the rhizosphere and phyllosphere., Conclusions: Our study revealed the rhizosphere as an overlooked hotspot for methanol consumption in temperate grasslands. We further identified unusual new but potentially relevant methanol utilizers besides well-known methylotrophs in the phyllosphere and rhizosphere. We did not observe a plant host-specific methanol utilizer community. Our results suggest that our approach using quantitative SIP and metagenomics may be useful in future field studies to link gross methanol consumption rates with the rhizosphere and phyllosphere microbiome., (© 2022. The Author(s).)

In computational biomechanics, two separate types of models have been used predominantly to enhance the understanding of the mechanisms of action of the lumbosacral spine (LSS): Finite element (FE) and musculoskeletal multibody (MB) models. To combine advantages of both models, hybrid FE-MB models are an increasingly used alternative. The aim of this paper is to develop, calibrate, and validate a novel passive hybrid FE-MB open-access simulation model of a ligamentous LSS using ArtiSynth. Based on anatomical data from the Male Visible Human Project, the LSS model is constructed from the L1-S1 rigid vertebrae interconnected with hyperelastic fiber-reinforced FE intervertebral discs, ligaments, and facet joints. A mesh convergence study, sensitivity analyses, and systematic calibration were conducted with the hybrid functional spinal unit (FSU) L4/5. The predicted mechanical responses of the FSU L4/5, the lumbar spine (L1-L5), and the LSS were validated against literature data from in vivo and in vitro measurements and in silico models. Spinal mechanical responses considered when loaded with pure moments and combined loading modes were total and intervertebral range of motions, instantaneous axes and centers of rotation, facet joint contact forces, intradiscal pressures, disc bulges, and stiffnesses. Undesirable correlations with the FE mesh were minimized, the number of crisscrossed collagen fiber rings was reduced to five, and the individual influences of specific anatomical structures were adjusted to in vitro range of motions. Including intervertebral motion couplings for axial rotation and nonlinear stiffening under increasing axial compression, the predicted kinematic and structural mechanics responses were consistent with the comparative data. The results demonstrate that the hybrid simulation model is robust and efficient in reproducing valid mechanical responses to provide a starting point for upcoming optimizations and extensions, such as with active skeletal muscles., Competing Interests: The authors have declared that no competing interests exist.

Problem: Student-preceptor discontinuity during 3rd-year clerkships limits the quality and quantity of supervision, teaching, and feedback. Although longitudinal integrative clerkships increase continuity and are associated with improved student and preceptor experience, they require schoolwide curricular reform. Alternative innovations enhancing student-preceptor relationships within the constraints of a traditional block clerkship may demonstrate similar benefits., Intervention: We piloted a continuity-enhanced general pediatric ambulatory schedule during 2 consecutive clerkship blocks in 2013. Students in the continuity-enhanced model (n = 29) were assigned 1-3 primary clinic preceptors, whereas those in the traditional model (n = 30) worked with 5-8. Data were gathered from student assessments and anonymous student and preceptor surveys. We used t and Fisher's exact tests to compare the two groups and performed thematic analysis of free-text survey comments., Context: Our school utilizes a block clerkship model with approximately 30 students rotating through the pediatric clerkship every 8 weeks. During the 3-week ambulatory portion, students spend 8 half days in the general pediatric ambulatory clinic. At the conclusion of each clinic, attendings completed brief student evaluation cards. Traditionally, student and attending schedules were created independently, resulting in transient supervisory relationships and dissatisfaction with clinical engagement, feedback, and evaluation., Outcome: Seventy-three percent (43/59) of the students completed the survey. Ten general ambulatory attendings collectively completed 87.5% (35/40) of the monthly surveys. Continuity students received significantly more narrative evaluation comments (10.6 vs. 5.8, p <. 001) from general ambulatory clinic attendings and were more likely to have at least one general ambulatory clinic attending endorse being able to provide meaningful feedback and evaluation (n = 29, 100% vs. n = 20, 66.7%, p <. 001). Continuity students were also more likely to endorse being able to ask at least one of these attendings for a letter of recommendation (71.4% vs. 9.1%, p <. 001) and to have at least one general ambulatory clinic attending endorse being able to provide a meaningful letter of recommendation if asked (62.1% vs. 3.3%, p <. 001). Students (88.4%) and attendings (85.7%) preferred the continuity-enhanced schedule. The most frequent theme of both student and attending free-text survey remarks were relationships and assessment., Lessons Learned: Intentional scheduling of clerkship students to enhance preceptor continuity resulted in significant positive outcomes echoing the relationship-based educational benefits of longitudinal clerkships, particularly in regards to student assessment and feedback. Clerkship directors and other medical educators should consider implementing small changes within block clerkships to maximize student-preceptor continuity.

Biological nitrogen fixation (BNF) is considered one of the key plant-growth-promoting (PGP) factors for diazotrophic organisms. Whether the iron and iron-molybdenum nitrogenases of Kosakonia radicincitans contribute to its PGP effect is yet to be proven. Hence, for the first time, we conducted site-directed mutagenesis in K. radicincitans to knock out anfH and (or) nifH as a mean to deactivate BNF in this strain. We used 15 N 2 -labeled air to trace BNF activities in ΔanfH, ΔnifH, and ΔanfHΔnifH mutants. Assessing bacterial growth, nitrogen content, and 15 N incorporation revealed that BNF is impaired in K. radicincitans DSM16656 T ΔnifH and ΔanfHΔnifH. However, we detected no significant contribution of the Fe nitrogenase to biological dinitrogen assimilation under our pure bacterial culture experimental conditions. Such nondiazotrophic K. radicincitans DSM16656 T mutants represent excellent tools for investigating nitrogen nutrition in K. radicincitans-inoculated plants.

Field experiments were conducted to test and evaluate the initial atmospheric carbon dioxide (CO 2 ) measurement capability of airborne, high-energy, double-pulsed, 2-μm integrated path differential absorption (IPDA) lidar. This IPDA was designed, integrated, and operated at the NASA Langley Research Center on-board the NASA B-200 aircraft. The IPDA was tuned to the CO 2 strong absorption line at 2050.9670 nm, which is the optimum for lower tropospheric weighted column measurements. Flights were conducted over land and ocean under different conditions. The first validation experiments of the IPDA for atmospheric CO 2 remote sensing, focusing on low surface reflectivity oceanic surface returns during full day background conditions, are presented. In these experiments, the IPDA measurements were validated by comparison to airborne flask air-sampling measurements conducted by the NOAA Earth System Research Laboratory. IPDA performance modeling was conducted to evaluate measurement sensitivity and bias errors. The IPDA signals and their variation with altitude compare well with predicted model results. In addition, off-off-line testing was conducted, with fixed instrument settings, to evaluate the IPDA systematic and random errors. Analysis shows an altitude-independent differential optical depth offset of 0.0769. Optical depth measurement uncertainty of 0.0918 compares well with the predicted value of 0.0761. IPDA CO 2 column measurement compares well with model-driven, near-simultaneous air-sampling measurements from the NOAA aircraft at different altitudes. With a 10-s shot average, CO 2 differential optical depth measurement of 1.0054±0.0103 was retrieved from a 6-km altitude and a 4-GHz on-line operation. As compared to CO 2 weighted-average column dry-air volume mixing ratio of 404.08 ppm, derived from air sampling, IPDA measurement resulted in a value of 405.22±4.15  ppm with 1.02% uncertainty and 0.28% additional bias. Sensitivity analysis of environmental systematic errors correlates the additional bias to water vapor. IPDA ranging resulted in a measurement uncertainty of <3  m.

Double-pulsed 2-μm integrated path differential absorption (IPDA) lidar is well suited for atmospheric CO 2 remote sensing. The IPDA lidar technique relies on wavelength differentiation between strong and weak absorbing features of the gas normalized to the transmitted energy. In the double-pulse case, each shot of the transmitter produces two successive laser pulses separated by a short interval. Calibration of the transmitted pulse energies is required for accurate CO 2 measurement. Design and calibration of a 2-μm double-pulse laser energy monitor is presented. The design is based on an InGaAs pin quantum detector. A high-speed photoelectromagnetic quantum detector was used for laser-pulse profile verification. Both quantum detectors were calibrated using a reference pyroelectric thermal detector. Calibration included comparing the three detection technologies in the single-pulsed mode, then comparing the quantum detectors in the double-pulsed mode. In addition, a self-calibration feature of the 2-μm IPDA lidar is presented. This feature allows one to monitor the transmitted laser energy, through residual scattering, with a single detection channel. This reduces the CO 2 measurement uncertainty. IPDA lidar ground validation for CO 2 measurement is presented for both calibrated energy monitor and self-calibration options. The calibrated energy monitor resulted in a lower CO 2 measurement bias, while self-calibration resulted in a better CO 2 temporal profiling when compared to the in situ sensor.

Background: Impairment of regulatory T cells (Tregs) in common autoimmune diseases seems likely. However, the extent of Treg deficiency (number, function) or differential susceptibility of T effector cells (Teffs) to suppression is not completely understood. We hypothesize that even in healthy individuals both cell populations are heterogeneous and differ in their suppressive capability and their susceptibility to suppression., Material and Methods: Lymphocytes were enriched by MACS for CD4(+)CD25(+) Tregs or CD4(+)CD25(-) Teffs. After multicolour staining (anti-CD25, anti-CD127, anti-CD49d or anti-CD45RA) highly purified Treg and Teff subpopulations were collected by FACS. Functional capacity of Tregs or suppressive susceptibility of Teffs was analyzed in an in vitro assay., Results: When CD4(+)CD25(high)CD127(-/low) CD49d(-) Tregs were tested on naive CD4(+)CD127(+)CD25(-)CD45RA(+) Teffs (93.8 %) suppression was almost complete, while the suppressive capacity of CD4(+)CD25(high)CD127(-/low) CD49d(+) Tregs was significantly less (71.8 %). Suppressive activity was low when CD4(+)CD25(high)CD127(-/low) CD49d(+) Tregs were analyzed on CD4(+)CD127(+)CD25(-)CD45RA(-) Teffs (48.7%)., Conclusion: Although CD49d(+) Tregs are functional, the suppressive capacity is significantly lower compared to CD49d(-) Tregs. CD45RA(+) Teffs can be completely suppressed, while CD45RA(-) Teffs display relative resistance. Phenotypic and functional heterogeneity of Tregs as well as Teffs has to be considered when analyzing deficiencies in immune regulation., (© 2014 S. Karger AG, Basel.)

Multiple sclerosis (MS) is an autoimmune disorder directed against self antigens of the central nervous system. CD4(+)CD25(+)FoxP3(+) regulatory T cell (T(reg)) mediated suppression is an essential mechanism of self-tolerance. We studied whether changes in the suppressive function of a mixture of CD25(high) and CD25(intemediate) expressing T(reg) cells in myelin basic protein (MBP)-induced proliferation occurred in untreated MS patients. Suppression of MBP-induced proliferation was observed in 13 out of 29 (45%) MS patients; this was significantly (p<0.05) less compared with 17 out of 19 (89%) healthy individuals. Relative T(reg) counts was significantly increased in MS patients (mean+/-S.D.; 20+/-8%) compared with healthy individuals (15+/-5%). These findings suggest that impaired T(reg) function may be involved in pathogenesis of MS.

Red cell membrane proteins are sequentially expressed during erythroid development and differentiation. Spectrins have already been synthesized in early erythroid precursors such as pronormoblasts, and band 3 (B3) appears at nearly the same stage. Protein 4.1 appears next, followed by protein 4.2 (P4.2) at the very late erythroblast stage. The methylation states of the promoter 5'-CG-3' sites are known to be linked to the regulation of promoter function by modulating DNA-protein interactions and the structure of chromatin. Hence, the genes for B3, P4.2, and beta-spectrin (beta-SP) appear to be suitable models to study the relationship between methylation of promoter 5'-CG-3' sites and the sequential expression of genes during human erythroid development and differentiation. We have examined methylation profiles in the promoter regions of the genes (ELB42, EPB3, and SPTB) for the human erythroid membrane proteins P4.2, B3, and beta-SP by applying the bisulfite genomic sequencing method. Our results demon strate the following: (1) The promoter regions of EPB3 and ELB42 are extensively methylated in DNA from human peripheral blood mononuclear cells, but the SPTB promoter is totally unmethylated. (2) During erythroid differentiation, DNA methylation patterns change as follows: (a) ELB42 is unmethylated in DNA from erythroid-committed blastic cells, such as the human cell lin UT-7/EPO, but is methylated in erythroblasts from peripheral blood burst-forming unit erythroid (BFU-E) in the second phase of the liquid-culture method. Messenger RNA (mRNA) from ELB42 is first detected in early erythroblasts, and P4.2 is expressed in late erythroblasts. (b) In contrast, EPB3 is consistently methylated in UT-7/EPO cells and in cultured erythroblasts from BFU-E from human peripheral blood. B3 mRNA and protein are already expressed in early erythroblasts. (c) SPTB remains unmethylated in human DNA from UT-7/EPO cells and from cultured erythroblasts. These results document the diversity of the reactions of human promoter sequences to the modulating influence of DNA methylation. Whereas the human SPTB promoter conforms to expectations in that it is unmethylated and fully active throughout erythroid development, high levels of promoter methylation correlate with promoter activity for the EPB3 and ELB42 genes during their sequential activation in erythrocyte differentiation.

The methylation state of 5'-CG-3' sites is known to be linked to the regulation of promoter function by modulating DNA-protein interactions and to the structure of chromatin. As part of a project to determine methylation patterns in the human genome, we examined the methylation profiles of several genes for human erythroid membrane proteins: ELB42 (protein 4.2), EPB3 (band 3), SPTB gene (beta-spectrin), and ANK1 (ankyrin). The bisulfite protocol of the genomic sequencing method was applied. The number of 5'-CG-3' dinucleotides was the most abundant in SPTB and ANK1, much less in EPB3, and the least in ELB42. In the DNA of peripheral blood mononuclear cells from healthy individuals, the promoter regions of EPB3 and ELB42 were extensively methylated, but the SPTB and ANK1 promoters were totally unmethylated. We also investigated methylation profiles in peripheral blood mononuclear cells from patients with red cell membrane diseases, such as complete protein 4.2 deficiency due to ELB42 mutations, hereditary spherocytosis with EPB3 mutations, and hereditary elliptocytosis with SPTB mutations. The DNA methylation states in these genes of erythroid cells, which we obtained at the second phase of the 2-phase liquid culture of erythroid precursor cells in the peripheral blood, were essentially identical or very similar to those of peripheral blood mononuclear cells. In disease states, the DNA methylation profiles of these red cell membrane protein genes were essentially not different from those in healthy individuals (statistically not significant).

A plant growth promoting bacterial isolate (D5/23T) from the phyllosphere of winter wheat, able to fix atmospheric nitrogen and to produce auxines and cytokinins was investigated in a polyphasic taxonomy approach. Phylogenetic analyses using the 16S rRNA gene sequence of the strain clearly indicated that the strain belonged to the family Enterobacteriaceae, most closely related to Enterobacter cloacae with 99.0% and Enterobacter dissolvens with 98.5% sequence similarity. Phylogenetic analysis derived from the sequence of the rpoB gene showed the highest sequence similarities to Enterobacter cowanii (93.0%) but supported the distinct position of strain D5/23T. The isolate produced a fatty acid pattern typical for members of the family Enterobacteriaceae. On the basis of the phylogenetic analyses, DNA-DNA hybridizations, and the unique physiological and biochemical characteristics, we propose that strain D5/23T represents a new species of the genus Enterobacter for which we propose the name Enterobacter radicincitans sp. nov.

The human nuclear 5'-(CGG)(n)-3'-binding protein (CGGBP1) influences the expression of the fragile X mental retardation (FMR1) gene by specifically interacting with the 5'-(CGG)(n>5)-3' repeat in its 5' untranslated region. Here, we show that CGGBP1 binds to 5'-(CGG)(n)-3' repeats with n>or=5 and to interrupted repeats. The genomic and mRNA organization of the human and murine CGGBP1 genes was studied and the human gene was mapped to chromosome 3p. Due to alternative polyadenylation, mRNAs of 1.2 and 4.5 kb are transcribed at varying ratios in human and murine cells and in embryonic, fetal, and adult tissues. The human and the murine genes, including promoters and large parts of the untranslated regions, are highly conserved. A sequence of 235 nucleotides 5' upstream of CGGBP1 is essential for promoter activity in transfection experiments. Complete in vitro methylation inactivates the promoter, which is unmethylated in human cells as shown by bisulfite genomic sequencing.

The intramuscular (i.m.) injection of human adenovirus type 12 (Ad12) into newborn Syrian hamsters caused widespread dissemination of up to 15 tumors over the entire peritoneal cavity in 70-90% of the animals within 30-50 days. Subcutaneous (s.c.) injections led to local tumor formation only. Independent of location, tumor histology revealed Homer-Wright rosette-like structures typical for primitive neuroectodermal tumors (PNET). All tumor cells showed markers indicative of neuroectodermal and mesenchymal derivations. Each Ad12-induced tumor cell carried multiple copies of integrated Ad12 genomes at one chromosomal site which was different for each tumor. For Ad12 tumor induction in hamsters, the patterns of Ad12 viral and cellular gene expression were important and were affected by changes in DNA methylation, both in the integrated Ad12 DNA and the cellular genome. By applying the bisulfite protocol, the de novo DNA methylation in the integrated Ad12 genomes was determined. These patterns were complex, characterized by regional initiation and by excluding genome segments in the E1A and E1B promoters. In all tumors, the Ad12 segments E1A, E1B, E2A, parts of E3 and E4 were similarly transcribed, as shown by the RT-PCR and DNA microarray methods. Changes in the transcription of a large number of cellular genes was assessed by using mouse gene microarrays encompassing about 1980 different mouse genes with 87-96% homology to hamster genes. Similarities and differences existed in the transcription of cellular genes of different functional classes among the different Ad12-induced tumors. These alterations in cellular gene transcription may be an important parameter in the oncogenic transformation by Ad12.

In an effort to identify psoriasis-associated genes, we compared gene expression in normal and psoriatic skin, using differential display RT-PCR technique. Sequence analysis of a 650-bp cDNA fragment (clone 110) that was highly up-regulated in lesional skin revealed homology to a noncoding cDNA (NICE-2). By subsequent cDNA cloning, using RNA from psoriatic skin, we have identified two alternatively spliced mRNA-isoforms (0.5 and 4.4 kb), which differ in composition of their untranslated regions. By sequence comparison, we have mapped the novel gene, named S100A15, to the S100 gene cluster within the epidermal differentiation complex (chromosome 1q21). Analysis of the deduced amino acid sequence revealed a protein of 101 amino acids containing two potential EF-hand motifs with high homology to the S100A7. Northern blot hybridization and semiquantitative RT-PCR analysis confirmed the S100A15 overexpression in psoriasis, showing different levels of expression of the S100A15 mRNA isoforms. In situ hybridization of the S100A15 revealed a markedly increased staining of basal and suprabasal epidermal layers of psoriatic skin compared with healthy tissue. Our data suggest an involvement of the novel S100A15 in epidermal differentiation and inflammation and might therefore be important for the pathogenesis of psoriasis and other diseases.

We have been interested in the consequences of foreign DNA insertion into established mammalian genomes and have initially studied this problem in adenovirus type 12 (Ad12)-transformed cells or in Ad12-induced hamster tumors. Since integrates are frequently methylated de novo, it appears that they might be modified by an ancient defense mechanism against foreign DNA. In cells transgenic for the DNA of Ad12 or for the DNA of bacteriophage lambda, changes in cellular methylation and transcription patterns have been observed. Thus, the insertion of foreign DNA can have important functional consequences that are not limited to the site of foreign DNA insertion. These findings appear to be relevant also for tumor biology and for the interpretation of data derived from experiments with transgenic organisms. For most animals, the main portal of entry for foreign DNA is the gastrointestinal tract. Large amounts of foreign DNA are regularly ingested with the supply of nutrients. Starting in 1987/1988, we have been investigating the fate of orally administered foreign DNA in mice. Naked DNA of bacteriophage M13 and the cloned gene for the green fluorescent protein (GFP) of Aequorea victoria have been used as test molecules. Moreover, the plant-specific gene for the ribulose-1,5-bisphosphate carboxylase (rubisco) has been followed in mice after feeding soybean leaves. At least transiently, food-ingested DNA can be traced to different organs and, after transplacental transfer, to fetuses and newborns. There is no evidence for germ line transmission or for the expression of orally administered GFP DNA.

We have investigated the consequences of foreign DNA insertions into the genomes of mammalian cells in transgenic cell lines, in adenovirus type 12 (Ad12)-transformed cells, in Ad12-induced tumor cells or in transgenic mice. We have reported previously on the de novo methylation of integrated foreign genomes and on extensive changes in cellular patterns of DNA methylation upon foreign DNA insertion. These studies have been extended and several independent methods have been applied to document these alterations in cellular DNA methylation and gene expression patterns in transgenic cell lines and in transgenic mice. These data are relevant for the mechanism of (viral) oncogenesis and for the interpretation of data gathered in experiments with transgenic animals.

The insertion of adenovirus type 12 (Ad12) DNA into the hamster genome and the transformation of these cells by Ad12 can lead to marked alterations in the levels of DNA methylation in several cellular genes and DNA segments. Since such alterations in DNA methylation patterns are likely to affect the transcription patterns of cellular genes, it is conceivable that these changes have played a role in the generation or the maintenance of the Ad12-transformed phenotype. We have now isolated clonal BHK21 hamster cell lines that carry in their genomes bacteriophage lambda and plasmid pSV2neo DNAs in an integrated state. Most of these cell lines contain one or multiple copies of integrated lambda DNA, which often colocalize with the pSV2neo DNA, usually in a single chromosomal site as determined by the fluorescent in situ hybridization technique. In different cell lines, the loci of foreign DNA insertion are different. The inserted bacteriophage lambda DNA frequently becomes de novo methylated. In some of the thus-generated hamster cell lines, the levels of DNA methylation in the retrotransposon genomes of the endogenous intracisternal A particles (IAP) are increased in comparison to those in the non-lambda-DNA-transgenic BHK21 cell lines. These changes in the methylation patterns of the IAP subclone I (IAPI) segment have been documented by restriction analyses with methylation-sensitive restriction endonucleases followed by Southern transfer hybridization and phosphorimager quantitation. The results of genomic sequencing experiments using the bisulfite protocol yielded additional evidence for alterations in the patterns of DNA methylation in selected segments of the IAPI sequences. In these experiments, the nucleotide sequences in >330 PCR-generated cloned DNA molecules were determined. Upon prolonged cultivation of cell lines with altered cellular methylation patterns, these differences became less apparent, perhaps due to counterselection of the transgenic cells. The possibility existed that the hamster BHK21 cell genomes represent mosaics with respect to DNA methylation in the IAPI segment. Hence, some of the cells with the patterns observed after lambda DNA integration might have existed prior to lambda DNA integration and been selected by chance. A total of 66 individual BHK21 cell clones from the BHK21 cell stock have been recloned up to three times, and the DNAs of these cell populations have been analyzed for differences in IAPI methylation patterns. None have been found. These patterns are identical among the individual BHK21 cell clones and identical to the patterns of the originally used BHK21 cell line. Similar results have been obtained with nine clones isolated from BHK21 cells mock transfected by the Ca2+-phosphate precipitation procedure with DNA omitted from the transfection mixture. In four clonal sublines of nontransgenic control BHK21 cells, genomic sequencing of 335 PCR-generated clones by the bisulfite protocol revealed 5'-CG-3' methylation levels in the IAPI segment that were comparable to those in the uncloned BHK21 cell line. We conclude that the observed changes in the DNA methylation patterns in BHK21 cells with integrated lambda DNA are unlikely to preexist or to be caused by the transfection procedure. Our data support the interpretation that the insertion of foreign DNA into a preexisting mammalian genome can alter the cellular patterns of DNA methylation, perhaps via changes in chromatin structure. The cellular sites affected by and the extent of these changes could depend on the site and size of foreign DNA insertion.

The insertion of foreign DNA into the genomes of established cells and organisms and the consequences of this integration event are of significance for viral oncology, reverse genetics, transgenic organisms, human somatic gene therapy and evolution. This review summarizes recent experimental findings and focuses on the alteration of cellular DNA methylation at regions remote from the site of insertion. We also discuss experimental data demonstrating that foreign DNA ingested by mice is not completely degraded in their gastrointestinal tracts; fragments of this DNA have been found to be covalently linked to DNA with 70% homology to the mouse IgE receptor gene.