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Root pressure, also manifested as profusive sap flowing from cut stems, is a phenomenon in some species that has perplexed biologists for much of the last century. It is associated with increased crop production under drought, but its function and regulation remain largely unknown. In this study, we investigated the initiation, mechanisms, and possible adaptive function of root pressure in six genotypes of Sorghum bicolor during a drought experiment in the greenhouse. We observed that root pressure was induced in plants exposed to drought followed by re-watering but possibly inhibited by 100% re-watering in some genotypes. We found that root pressure in drought stressed and re-watered plants was associated with greater ratio of fine: coarse root length and shoot biomass production, indicating a possible role of root allocation in creating root pressure and adaptive benefit of root pressure for shoot biomass production. Using RNA-Seq, we identified gene transcripts that were up- and down-regulated in plants with root pressure expression, focusing on genes for aquaporins, membrane transporters, and ATPases that could regulate inter- and intra-cellular transport of water and ions to generate positive xylem pressure in root tissue., 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 © 2021 Drobnitch, Comas, Flynn, Ibarra Caballero, Barton, Wenz, Person, Bushey, Jahn and Gleason.)

Common wheat ( Triticum aestivum L.) is a global staple crop, and insect pests can impact grain yield. The wheat stem sawfly ( Cephus cinctus , WSS) is a major wheat pest, and while partial resistance has been deployed by breeding for a solid-stem trait, this trait is affected by environment. Here, a proteomics and metabolomics study was performed on four wheat cultivars to characterize a molecular response to WSS infestation. The cultivars Hatcher (hollow-stem partially tolerant), Conan (semisolid-stem-resistant), and Denali and Reeder (hollow-stem-susceptible) were infested with WSS, and changes in stem proteins and metabolites were characterized using liquid chromatography-mass spectrometry. The proteome was characterized as 1830 proteins that included five major biological processes, including metabolic processes and response to stimuli, and the metabolome (1823 metabolites) spanned eight chemical superclasses, including alkaloids, benzenoids, and lipids. All four varieties had a molecular response to WSS following infestation. Hatcher had the most distinct changes, whereby 62 proteins and 29 metabolites varied in metabolic pathways involving enzymatic detoxification, proteinase inhibition, and antiherbivory compound production via benzoxazinoids, neolignans, and phenolics. Taken together, these data demonstrate variation in the wheat stem molecular response to WSS infestation and support breeding for molecular resistance in hollow-stem cultivars.

Objective: Rhizoctonia solani is a soil-borne fungal pathogen of many important crop plants. In rice, R. solani causes sheath blight disease, which results in devastating grain yield and quality losses. Few methods are available to control this pathogen and classic single gene resistance mechanisms in rice plants have not been identified. We hypothesize that alternate means of control are available in the environment including free-living amoebae. Amoebae are soil-, water- and air-borne microorganisms that are predominantly heterotrophic. Many amoeba species are mycophagous, and several harm their prey using mechanisms other than phagocytosis. Here, we used light and scanning electron microscopy to survey the interactions of R. solani with four amoeba species, with the goal of identifying amoebae species with potential for biocontrol., Results: We observed a wide range of responses during interactions of R. solani with four different free-living amoebae. Two Acanthamoeba species encyst in co-cultures with R. solani at higher rates than medium without R. solani. Vermamoeba vermiformis (formerly Hartmanella vermiformis) attach to R. solani mycelium and are associated with mycelial shriveling and perforations of fungal cell walls, indicating an antagonistic interaction. No phenotypic changes were observed in co-cultures of Dictyostelium discoideum and R. solani.

Produced water (PW) from oil and gas operations is considered a potential resource for food crop irrigation because of increasing water scarcity in dryland agriculture. However, efforts to employ PW for agriculture have been met with limited success. A greenhouse study was performed to evaluate the effects of PW on physiological and morphological traits of spring wheat (Triticum aestivum). Plants were irrigated with water treatments containing 10 and 50% PW (PW10 and PW50, respectively) and compared to a matching 50% salinity (NaCl50) and 100% tap water controls. Compared to controls, plants watered with PW10 and PW50 exhibited developmental arrest and reductions in aboveground and belowground biomass, photosynthetic efficiency, and reproductive growth. Decreases in grain yield ranged from 70 to 100% in plants irrigated with PW compared to the tap water control. Importantly, the PW10 and NaCl50 treatments were comparable for morphophysiological effects, even though NaCl50 contained 5 times the total dissolved solids, suggesting that constituents other than NaCl in PW contributed to plant stress. These findings indicate that despite discharge and reuse requirements focused on total dissolved solids, salinity stress may not be the primary factor affecting crop health. The results of the present study are informative for developing guidelines for the use of PW in agriculture to ensure minimal effects on crop morphology and physiology. Environ Toxicol Chem 2019;38:1756-1769. © 2019 SETAC., (© 2019 SETAC.)

Plant development, growth, and adaptation to stress are regulated by phytohormones, which can influence physiology even at low concentrations. Phytohormones are chemically grouped according to both structure and function as auxins, cytokinins, abscisic acid, jasmonates, salicylates, gibberellins, and brassinosteroids, among others. This chemical diversity and requirement for highly sensitive detection in complex matrices create unique challenges for comprehensive phytohormone analysis. Here, we present a robust and efficient quantitative UPLC-MS/MS assay for 17 phytohormones, including jasmonates, salicylates, abscisic acid, gibberellins, cytokinins, and auxins. Using this assay, 12 phytohormones were detected and quantified in sorghum plant tissue without the need for solid phase extraction (SPE) or liquid-liquid extraction. Variation of phytohormone profiles was explored in both root and leaf tissues between three genotypes, harvested at two different developmental time points. The results highlight the importance of tissue type, sampling time, and genetic factors when designing experiments that involve phytohormone analysis of sorghum. This research lays the groundwork for future studies, which can combine phytohormone profiling with other datasets such as transcriptome, soil microbiome, genome, and metabolome data, to provide important functional information about adaptation to stress and other environmental variables.

Root exudation is an important plant process by which roots release small molecules into the rhizosphere that serve in overall plant functioning. Yet, there is a major gap in our knowledge in translating plant root exudation in artificial systems (i.e., hydroponics, sterile media) to crops, specifically for soils expected in field conditions. Sorghum ( Sorghum bicolor L. Moench) root exudation was determined using both ultra-performance liquid chromatography and gas chromatography mass spectrometry-based non-targeted metabolomics to evaluate variation in exudate composition of two sorghum genotypes among three substrates (sand, clay, and soil). Above and belowground plant traits were measured to determine the interaction between sorghum genotype and belowground substrate. Plant growth and quantitative exudate composition were found to vary largely by substrate. Two types of changes to rhizosphere metabolites were observed: rhizosphere-enhanced metabolites (REMs) and rhizosphere-abated metabolites (RAMs). More REMs and RAMs were detected in sand and clay substrates compared to the soil substrate. This study demonstrates that belowground substrate influences the root exudate profile in sorghum, and that two sorghum genotypes exuded metabolites at different magnitudes. However, metabolite identification remains a major bottleneck in non-targeted metabolite profiling of the rhizosphere., Competing Interests: The authors declare no conflict of interest.

Plant physiology and metabolism are important components of a plant response to microbial pathogens. Physiological resistance of common bean (Phaseolus vulgaris L.) to the fungal pathogen Sclerotinia sclerotiorum has been established, but the mechanisms of resistance are largely unknown. Here, the physiological and metabolic responses of bean varieties that differ in physiological resistance to S. sclerotiorum are investigated. Upon infection, the resistant bean variety A195 had a unique physiological response that included reduced photosynthesis and maintaining a higher leaf surface pH during infection. Leaf metabolomics was performed on healthy tissue adjacent to the necrotic lesion at 16, 24, and 48 hr post inoculation, and 144 metabolites were detected that varied between A195 and Sacramento following infection. The metabolites that varied in leaves included amines/amino acids, organic acids, phytoalexins, and ureides. The metabolic pathways associated with resistance included amine metabolism, uriede-based nitrogen remobilization, antioxidant production, and bean-specific phytoalexin production. A second experiment was conducted in stems of 13 bean genotypes with varying resistance. Stem resistance was associated with phytoalexin production, but unlike leaf metabolism, lipid changes were associated with susceptibility. Taken together, the data supports a multifaceted, physiometabolic response of common bean to S. sclerotiorum that mediates resistance., (© 2018 John Wiley & Sons Ltd.)

Background: Free-living amoebae (FLA) are voracious feeders, consuming bacteria and other microbes during colonization of the phytobiome. FLA are also known to secrete bacteriocidal or bacteriostatic compounds into their growth environment., Methodology and Principal Findings: Here, we explore the impacts of co-cultivation of five FLA species, including Acanthamoeba castellanii, A. lenticulata, A. polyphaga, Dictyostelium discoideum and Vermamoeba vermiformis, on survival of two devastating bacterial pathogens of rice, Xanthomonas oryzae pathovars (pv.) oryzae and oryzicola. In co-cultivation assays, the five FLA species were either bacteriostatic or bactericidal to X. oryzae pv. oryzae and X. oryzae pv. oryzicola. Despite these effects, bacteria were rarely detected inside amoebal cells. Furthermore, amoebae did not disrupt X. oryzae biofilms. The bactericidal effects persisted when bacteria were added to a cell-free supernatant from amoebal cultures, suggesting some amoebae produce an extracellular bactericidal compound., Conclusions/significance: This work establishes novel, basal dynamics between important plant pathogenic bacteria and diverse amoebae, and lays the framework for future mechanistic studies., Competing Interests: The authors have declared that no competing interests exist.

Wheat ( Triticum aestivum L.) is an important food crop, and biotic and abiotic stresses significantly impact grain yield. Wheat leaf and stem surface waxes are associated with traits of biological importance, including stress resistance. Past studies have characterized the composition of wheat cuticular waxes, however protocols can be relatively low-throughput and narrow in the range of metabolites detected. Here, gas chromatography-mass spectrometry (GC-MS) metabolomics methods were utilized to provide a comprehensive characterization of the chemical composition of cuticular waxes in wheat leaves and stems. Further, waxes from four wheat cultivars were assayed to evaluate the potential for GC-MS metabolomics to describe wax composition attributed to differences in wheat genotype. A total of 263 putative compounds were detected and included 58 wax compounds that can be classified (e.g., alkanes and fatty acids). Many of the detected wax metabolites have known associations to important biological functions. Principal component analysis and ANOVA were used to evaluate metabolite distribution, which was attributed to both tissue type (leaf, stem) and cultivar differences. Leaves contained more primary alcohols than stems such as 6-methylheptacosan-1-ol and octacosan-1-ol. The metabolite data were validated using scanning electron microscopy of epicuticular wax crystals which detected wax tubules and platelets. Conan was the only cultivar to display alcohol-associated platelet-shaped crystals on its abaxial leaf surface. Taken together, application of GC-MS metabolomics enabled the characterization of cuticular wax content in wheat tissues and provided relative quantitative comparisons among sample types, thus contributing to the understanding of wax composition associated with important phenotypic traits in a major crop., Competing Interests: The authors declare no conflict of interest.

Iron (Fe) is an essential element for plants, utilized in nearly every cellular process. Because the adjustment of uptake under Fe limitation cannot satisfy all demands, plants need to acclimate their physiology and biochemistry, especially in their chloroplasts, which have a high demand for Fe. To investigate if a program exists for the utilization of Fe under deficiency, we analyzed how hydroponically grown Arabidopsis ( Arabidopsis thaliana ) adjusts its physiology and Fe protein composition in vegetative photosynthetic tissue during Fe deficiency. Fe deficiency first affected photosynthetic electron transport with concomitant reductions in carbon assimilation and biomass production when effects on respiration were not yet significant. Photosynthetic electron transport function and protein levels of Fe-dependent enzymes were fully recovered upon Fe resupply, indicating that the Fe depletion stress did not cause irreversible secondary damage. At the protein level, ferredoxin, the cytochrome- b 6 f complex, and Fe-containing enzymes of the plastid sulfur assimilation pathway were major targets of Fe deficiency, whereas other Fe-dependent functions were relatively less affected. In coordination, SufA and SufB, two proteins of the plastid Fe-sulfur cofactor assembly pathway, were also diminished early by Fe depletion. Iron depletion reduced mRNA levels for the majority of the affected proteins, indicating that loss of enzyme was not just due to lack of Fe cofactors. SufB and ferredoxin were early targets of transcript down-regulation. The data reveal a hierarchy for Fe utilization in photosynthetic tissue and indicate that a program is in place to acclimate to impending Fe deficiency., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Metabolomics is an emerging method to improve our understanding of how genetic diversity affects phenotypic variation in plants. Recent studies have demonstrated that genotype has a major influence on biochemical variation in several types of plant tissues, however, the association between metabolic variation and variation in morphological and physiological traits is largely unknown. Sorghum bicolor (L.) is an important food and fuel crop with extensive genetic and phenotypic variation. Sorghum lines have been bred for differing phenotypes beneficial for production of grain (food), stem sugar (food, fuel), and cellulosic biomass (forage, fuel), and these varying phenotypes are the end products of innate metabolic programming which determines how carbon is allocated during plant growth and development. Further, sorghum has been adapted among highly diverse environments. Because of this geographic and phenotypic variation, the sorghum metabolome is expected to be highly divergent; however, metabolite variation in sorghum has not been characterized. Here, we utilize a phenotypically diverse panel of sorghum breeding lines to identify associations between leaf metabolites and morpho-physiological traits. The panel (11 lines) exhibited significant variation for 21 morpho-physiological traits, as well as broader trends in variation by sorghum type (grain vs. biomass types). Variation was also observed for cell wall constituents (glucan, xylan, lignin, ash). Non-targeted metabolomics analysis of leaf tissue showed that 956 of 1181 metabolites varied among the lines (81%, ANOVA, FDR adjusted p < 0.05). Both univariate and multivariate analyses determined relationships between metabolites and morpho-physiological traits, and 384 metabolites correlated with at least one trait (32%, p < 0.05), including many secondary metabolites such as glycosylated flavonoids and chlorogenic acids. The use of metabolomics to explain relationships between two or more morpho-physiological traits was explored and showed chlorogenic and shikimic acid to be associated with photosynthesis, early plant growth and final biomass measures in sorghum. Taken together, this study demonstrates the integration of metabolomics with morpho-physiological datasets to elucidate links between plant metabolism, growth, and architecture.

A major proportion of plant biomass is derived from the activity of the cambium, a lateral meristem responsible for vascular tissue formation and radial organ enlargement in a process termed secondary growth. In contrast to our relatively good understanding of the regulation of primary meristems, remarkably little is known concerning the mechanisms controlling secondary growth, particularly how cambial cell divisions are regulated and integrated with vascular differentiation. A genetic loss-of-function approach was used here to reveal a rate-limiting role for the Arabidopsis CYCLIN D3 (CYCD3) subgroup of cell-cycle genes in the control of cambial cell proliferation and secondary growth, providing conclusive evidence of a direct link between the cell cycle and vascular development. It is shown that all three CYCD3 genes are specifically expressed in the cambium throughout vascular development. Analysis of a triple loss-of-function CYCD3 mutant revealed a requirement for CYCD3 in promoting the cambial cell cycle since mutant stems and hypocotyls showed a marked reduction in diameter linked to reduced mitotic activity in the cambium. Conversely, loss of CYCD3 provoked an increase in xylem cell size and the expression of differentiation markers, showing that CYCD3 is required to restrain the differentiation of xylem precursor cells. Together, our data show that tight control of cambial cell division through developmental- and cell type-specific regulation of CYCD3 is required for normal vascular development, constituting part of a novel mechanism controlling organ growth in higher plants., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Bioenergy will be one component of a suite of alternatives to fossil fuels. Effective conversion of biomass to energy will require the careful pairing of advanced conversion technologies with biomass feedstocks optimized for the purpose. Lignocellulosic biomass can be converted to useful energy products via two distinct pathways: enzymatic or thermochemical conversion. The thermochemical pathways are reviewed and potential biotechnology or breeding targets to improve feedstocks for pyrolysis, gasification, and combustion are identified. Biomass traits influencing the effectiveness of the thermochemical process (cell wall composition, mineral and moisture content) differ from those important for enzymatic conversion and so properties are discussed in the language of biologists (biochemical analysis) as well as that of engineers (proximate and ultimate analysis). We discuss the genetic control, potential environmental influence, and consequences of modification of these traits. Improving feedstocks for thermochemical conversion can be accomplished by the optimization of lignin levels, and the reduction of ash and moisture content. We suggest that ultimate analysis and associated properties such as H:C, O:C, and heating value might be more amenable than traditional biochemical analysis to the high-throughput necessary for the phenotyping of large plant populations. Expanding our knowledge of these biomass traits will play a critical role in the utilization of biomass for energy production globally, and add to our understanding of how plants tailor their composition with their environment.

Dickeya dadantii is a plant-pathogenic bacterium that produces cellulose-containing biofilms, called pellicles, at the air-liquid interface of liquid cultures. D. dadantii pellicle formation appears to be an emergent property dependent upon at least three gene clusters, including cellulose synthesis, type III secretion system (T3SS) and flagellar genes. The D. dadantii cellulose synthesis operon is homologous to that of Gluconacetobacter xylinus, which is used for industrial cellulose production, and the cellulose nanofibres produced by D. dadantii were similar in diameter and branching pattern to those produced by G. xylinus. Salmonella enterica, an enterobacterium closely related to D. dadantii, encodes a second type of cellulose synthesis operon, and it produced biofilm strands that differed in width and branching pattern from those of D. dadantii and G. xylinus. Unlike any previously described cellulose fibre, the D. dadantii cellulose nanofibres were decorated with bead-like structures. Mutation of the cellulose synthesis operon genes resulted in loss of cellulose synthesis and production of a cellulase-resistant biofilm. Mutation of other genes required for pellicle formation, including those encoding FliA (a sigma factor that regulates flagella production), HrpL (a sigma factor that regulates the T3SS), and AdrA, a GGDEF protein, affected both biofilm and cell morphology. Mutation of the cellulose synthase bcsA or of bcsC resulted in decreased accumulation of the T3SS-secreted protein HrpN.

Biofuels provide a promising route of producing energy while reducing reliance on petroleum. Developing sustainable liquid fuel production from cellulosic feedstock is a major challenge and will require significant breeding efforts to maximize plant biomass production. Our approach to elucidating genes and genetic pathways that can be targeted for improving biomass production is to exploit the combination of genomic tools and genetic diversity in rice (Oryza sativa). In this study, we analyzed a diverse set of 20 recently resequenced rice varieties for variation in biomass traits at several different developmental stages. The traits included plant size and architecture, aboveground biomass, and underlying physiological processes. We found significant genetic variation among the 20 lines in all morphological and physiological traits. Although heritability estimates were significant for all traits, heritabilities were higher in traits relating to plant size and architecture than for physiological traits. Trait variation was largely explained by variety and breeding history (advanced versus landrace) but not by varietal groupings (indica, japonica, and aus). In the context of cellulosic biofuels development, cell wall composition varied significantly among varieties. Surprisingly, photosynthetic rates among the varieties were inversely correlated with biomass accumulation. Examining these data in an evolutionary context reveals that rice varieties have achieved high biomass production via independent developmental and physiological pathways, suggesting that there are multiple targets for biomass improvement. Future efforts to identify loci and networks underlying this functional variation will facilitate the improvement of biomass traits in other grasses being developed as energy crops.

We compare genome sequences of three closely related soft-rot pathogens that vary in host range and geographical distribution to identify genetic differences that could account for lifestyle differences. The isolates compared, Pectobacterium atrosepticum SCRI1043, P. carotovorum WPP14, and P. brasiliensis 1692, represent diverse lineages of the genus. P. carotovorum and P. brasiliensis genome contigs, generated by 454 pyrosequencing ordered by reference to the previously published complete circular chromosome of P. atrosepticum genome and each other, account for 96% of the predicted genome size. Orthologous proteins encoded by P. carotovorum and P. brasiliensis are approximately 95% identical to each other and 92% identical to P. atrosepticum. Multiple alignment using Mauve identified a core genome of 3.9 Mb conserved among these Pectobacterium spp. Each core genome is interrupted at many points by species-specific insertions or deletions (indels) that account for approximately 0.9 to 1.1 Mb. We demonstrate that the presence of a hrpK-like type III secretion system-dependent effector protein in P. carotovorum and P. brasiliensis and its absence from P. atrosepticum is insufficient to explain variability in their response to infection in a plant. Additional genes that vary among these species include those encoding peptide toxin production, enzyme production, secretion proteins, and antibiotic production, as well as differences in more general aspects of gene regulation and metabolism that may be relevant to pathogenicity.

The genome sequence of the Enterobacteriaceae phytopathogen Dickeya dadantii (formerly Erwinia chrysanthemi) revealed homologs of genes required for a complete flagellar secretion system and one flagellin gene. We found that D. dadantii was able to swim and swarm but that ability to swarm was dependent upon both growth media and temperature. Mutation of the D. dadantii fliA gene was pleiotropic, with the alternate sigma factor required for flagella production and development of disease symptoms but not bacterial growth in Nicotiana benthamiana leaves. The flagellar sigma factor was also required for multiple bacterial phenotypes, including biofilm formation in culture, bacterial adherence to plant tissue, and full expression of pectate lyase activity (but not cellulase or protease activity). Surprisingly, mutation of fliA resulted in the increased expression of avrL (a gene of unknown function in D. dadantii) and two pectate lyase gene homologs, pelX and ABF-0019391. Because FliA is a key contributor to virulence in D. dadantii, it is a new target for disease control.

RNA integrity is critical for successful RNA quantitation for mammalian tissues, but the level of integrity required differs among tissues. The level of integrity required for quantitation has not been determined for bacterial RNA. Three RNA isolation methods were evaluated for their ability to produce high quality RNA from Dickeya dadantii, a bacterium refractory to RNA isolation. Bacterial lysis with Trizol using standard protocols consistently gave low RNA yields with this organism. Higher yields due to improved bacterial cells lysis was achieved with an added hot SDS incubation step, but RNA quality was low as determined by the RNA Integrity Number (RIN). Contaminating DNA remained a problem with the hot SDS-Trizol method; RNA samples required repeated, rigorous DNase treatments to reduce DNA contamination to levels sufficient for successful real-time qRT-PCR. A hot SDS-hot phenol RNA method gave the highest RNA quality and required only two DNase treatments to remove DNA. The assessment of RNA integrity using the Agilent 2100 BioAnalyzer was critical for obtaining meaningful gene expression data. RIN values below 7.0 resulted in high variation and loss of statistical significance when gene expression was analyzed by real-time qRT-PCR. We found that RNA preparations of different quality yielded drastic differences in relative gene expression ratios and led to major errors in the quantification of transcript levels. This work provides guidelines for RNA isolation and quality assessment that will be valuable for gene expression studies in a wide range of bacteria.

Numerous salmonellosis outbreaks have been associated with vegetables, in particular sprouted seed. Thin aggregative fimbriae (Tafi), a component of the extracellular matrix responsible for multicellular behavior, are important for Salmonella enterica attachment and colonization of plants. Here, we demonstrate that the other surface polymers composing the extracellular matrix, cellulose, and O-antigen capsule also play a role in colonization of plants. Mutations in bacterial cellulose synthesis (bcsA) and O-antigen capsule assembly and translocation (yihO) reduced the ability to attach to and colonize alfalfa sprouts. A colanic acid mutant was unaffected in plant attachment or colonization. Tafi, cellulose synthesis, and O-antigen capsule, all of which contribute to attachment and colonization of plants, are regulated by AgfD, suggesting that AgfD is a key regulator for survival outside of hosts of Salmonella spp. The cellulose biosynthesis regulator adrA mutant was not affected in the ability to attach to or colonize plants; however, promoter probe assays revealed expression by cells attached to alfalfa sprouts. Furthermore, quantitative reverse-transcriptase polymerase chain reaction revealed differential expression of agfD and adrA between planktonic and plant-attached cells. In addition, there was no correlation among mutants between biofilm formation in culture and attachment to plants. Outside of animal hosts, S. enterica appears to rely on an arsenal of adhesins to persist on plants, which can act as vectors and perpetuate public health concerns.

Purpose: To examine the tolerance and mechanical function of an adjustable intraocular lens (IOL) in rabbit eyes., Methods: Implantation of the *Acri.Tec AR-1 PC/IOL into 14 rabbit eyes. Manipulation of the lens 8 weeks after implantation in order to change the refractive power. Follow-up for up to 5 months. Histopathologic examination of the eyes., Results: Implantation and mechanical adjustment of the PC/IOL were possible. Eyes healed normally. No difference between eyes containing the *Acri.Tec AR-1 PC/IOL and eyes containing the control PC/IOL could be detected with respect to signs of inflammatory reaction, corneal transparency, intraocular pressure and histopathologic appearance. Histopathologic examination of the eyes showed that the *Acri.Tec AR-1 PC/IOL did not cause any damage in rabbit eyes., Conclusion: The *Acri.Tec AR-1 PC/IOL is well tolerated in rabbit eyes for extended periods of time, suggesting that this PC/IOL should be well tolerated in the long run. Surgical adjustment of the adjusting element can be performed with little effort several weeks after implantation., (Copyright (c) 2006 S. Karger AG, Basel.)

Purpose: To report the safety of a newly developed intraocular lens (PC/IOL) with reversibly adjustable refractive power (*Acri.Tec AR-1 PC/IOL) after implantation into adult human eyes. The surgical setup and specially designed instruments required for implantation will be evaluated. The examined parameters included: stability of the IOL during implantation, positioning of the IOL, rotation of the IOL, integrity of the posterior lens capsule, central visual acuity, morphology of the eye, clinical signs of inflammation, intraocular pressure, refraction., Methods: The study was carried out on 35 human eyes after using the Acri.Tec AR-1 PC/IOL after lens removal through phacoemulsification; as control, the implantation of conventional PC/IOL into the second eye of 29 patients was used. The mean follow-up was 9 + 6 months. An initial adjustment surgery of the *Acri.Tec AR-1 PC/IOL was carried out 2 weeks after implantation in 2 patients; the follow-up of these eyes was 3 and 6 months., Results: Implantation was possible without any problem. Eyes healed normally. No difference between eyes containing the *Acri.Tec AR-1 PC/IOL and eyes containing conventional PC/IOLs could be detected with respect to visual acuity, stabilization of refractive error, signs of inflammatory reaction, slitlamp microscopic aspect of the eye and intraocular pressure. As intended, initial adjustment surgery successfully changed the mydriatic refraction from +1.0 resp. +0.75 dpt to 0 resp. -0.5 dpt. 26 bilaterally pseudophakic patients including 2 patients having had adjustment surgery had more than 3 months follow-up. Mean visual acuity in the AR-1 PC/IOL group at 3 months (0.76 +/-0.24) did not differ from mean visual acuity in the pseudophakic control group (0.74 +/-0.23). Adjusted eyes had a visual acuity of 1.0. No adverse effects occurred., Conclusion: Initial data suggest that the *Acri.Tec AR-1 PC/IOL can be safely implanted into the capsular bag of adult human eyes and can beadjusted after implantation, thereby achieving the desired change of refraction. Further exploration of the potential usefulness of this type of reversibly adjustable PC/IOL as a tool for refractive optimization is warranted., (Copyright 2005 S. Karger AG, Basel.)

Because archaea are generally associated with extreme environments, detection of nonthermophilic members belonging to the archaeal division Crenarchaeota over the last decade was unexpected; they are surprisingly ubiquitous and abundant in nonextreme marine and terrestrial habitats. Metabolic characterization of these nonthermophilic crenarchaeotes has been impeded by their intractability toward isolation and growth in culture. From studies employing a combination of cultivation and molecular phylogenetic techniques (PCR-single-strand conformation polymorphism, sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, and real-time PCR), we present evidence here that one of the two dominant phylotypes of Crenarchaeota that colonizes the roots of tomato plants grown in soil from a Wisconsin field is selectively enriched in mixed cultures amended with root extract. Clones recovered from enrichment cultures were found to group phylogenetically with sequences from clade C1b.A1. This work corroborates and extends our recent findings, indicating that the diversity of the crenarchaeal soil assemblage is influenced by the rhizosphere and that mesophilic soil crenarchaeotes are found associated with plant roots, and provides the first evidence for growth of nonthermophilic crenarchaeotes in culture.

Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of extracellular polysaccharide, cellulose, and protein filaments, known as curli. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here, we demonstrate that Erwinia chrysanthemi, which does not carry curli genes, requires the TTSS for pellicle formation. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found that hrpY, which encodes a two-component system response regulator homolog, is required for activity of hrpS, which encodes a sigma54-dependent enhancer-binding protein homolog. In turn, hrpS is required for activity of the sigma factor homolog hrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36 degrees C, even though TTSS genes were expressed at this temperature. We found that cellulose is a component of the E. chrysanthemi pellicle but that pellicle formation still occurs in a strain with an insertion in a cellulose synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors.

Background: Diabetic macular edema (DME) is the leading cause of severe visual loss in patients with diabetic retinopathy. This is so despite the fact that argon laser photocoagulation of the macula (M-ALC) has been shown to be beneficial. Recently, it has been suggested that pars plana vitrectomy (PPV) can lead to the resolution of DME and stop the deterioration of central visual acuity., Purpose: To explore the potential benefit of PPV for the treatment of DME., Patients and Methods: PPV was carried out in 30 eyes of 21 consecutive patients (median age 71 years, range 61-88 years) with type II diabetes mellitus suffering from DME. 23 eyes had non-proliferative diabetic retinopathy (NPDR) and 7 eyes had proliferative diabetic retinopathy (PDR) in addition to DME. Posterior vitreous detachment had to be carried out in all cases. If epiretinal membranes were present (23 eyes), they were removed. In 13 eyes (initially 11 eyes) the internal limiting membrane (ILM) was also removed. Prior to PPV 8 eyes had received M-ALC. Three eyes had M-ALC after PPV. One eye developed a retinal detachment 6 weeks after PPV and was excluded form the analysis. After an initial treatment failure two eyes underwent repeat PPV with peeling of the ILM. Both eyes of another patient had 2 repeat PPVs because of recurrent vitreous hemorrhage. Median follow-up was 16 months (range 1-62 months)., Results: Following PPV the macula flattened or became attached in 20/27 (74%) eyes. 15/18 (83%) eyes showed reduction or disappearance of leakage during fluorescein-angiography. Central visual acuity increased by two to six lines in 15/27 (56%) for the whole group at 6 months after PPV. For the subgroup (18 eyes) for which the evolution of visual acuity prior to PPV could be documented mean and median visual acuity had decreased markedly from 0.26 +/- 0.19 resp. 0.2 (range 0.03-0.6) to 0.12 +/- 0.09 resp. 0.1 (range 0.02-0.4) during the 12 months preceding PPV and increased to 0.28 +/- 0.23 resp. 0.2 (range 0.03-0.8) during the 12 months following PPV., Conclusion: PPV almost always results in a reduction and often complete disappearance of DME as evidenced by ophthalmoscopy and fluorescein-angiography. Most importantly, central visual acuity often increases, sometimes to a very large extent with dramatic improvement in quality of life of the patients., (Copyright (c) 2004 S. Karger AG, Basel.)

Purpose: To evaluate whether it is technically feasible and safe to implant and adjust an intraocular lens (IOL) with reversibly adjustable refractive power designed to correct residual postoperative refractive error., Setting: Animal study facility, Klinikum Mannheim, University of Heidelberg, Heidelberg, Germany., Methods: An Acri.Tec AR-1 posterior chamber IOL (PC IOL) was implanted in pig cadaver eyes and in rabbit eyes after the crystalline lens was removed by phacoemulsification. The IOL was manipulated to change the refractive power. The outcome measures were the stability of the IOL during implantation, IOL positioning and rotation, the ability to move the adjustment device after the IOL had been implanted for several weeks, clinical signs of inflammation, and changes in the histopathologic appearance of the eye., Results: Implantation and adjustment of refractive power were possible. The eyes healed normally. There was no difference between eyes with the AR-1 PC IOL and eyes with a control IOL in inflammatory reaction, corneal transparency, or histopathologic appearance., Conclusion: The AR-1 PC IOL was easy to implant and well tolerated in rabbit eyes. Surgical adjustment of the adjusting element was performed with little effort several weeks after implantation of the IOL.

Purpose: To determine the incidence of retinal detachment (RD) after neodymium:YAG (Nd:YAG) laser capsulotomy for posterior capsule opacification (PCO) and define the characteristics of this type of RD., Setting: A private practice, with statistical evaluation at a university department., Methods: Five hundred twenty-six consecutive eyes that had Nd:YAG capsulotomy because of visually disturbing PCO were followed prospectively for up to 52 months (median 21 months) to determine the incidence of RD. The median age of the 142 men and 320 women was 76 years and the median axial length, 23.3 mm (range 20.5 to 31.3 mm). Before the capsulotomy, all eyes had uneventful phacoemulsification with implantation of a posterior chamber intraocular lens (PC IOL). Eyes with additional surgical procedures or with panretinal laser photocoagulation were excluded. Eyes in which a retinal hole had been treated before Nd:YAG (n = 4) were included. The fundus was examined by binocular indirect ophthalmoscopy with a 2.2 diopter Volk lens in full mydriasis. If necessary, an examination with a 3-mirror lens was done., Results: The incidence of RD was 0% (0/483; 95% confidence interval [CI], 0.0%-0.8%) at 6 months, 0% (0/407; 95% CI, 0.0%-0.9%) at 12 months, and 0.5% (1/213; 95% CI, 0.0%-2.6%) at 24 months. Twenty-one months after Nd:YAG capsulotomy, 1 eye developed an RD that occurred as the result of a horseshoe tear that reopened. The tear, which formed 7 months after phacoemulsification and PC IOL implantation and 6 years before Nd:YAG capsulotomy, had been immediately treated with argon laser photocoagulation. Excluding the 4 eyes with preexisting tears, presumably sealed retinal holes, the incidence at 24 months was 0% (0/212; 95% CI, 0.0%-1.7%). No other eye developed RD over the follow-up., Conclusions: Retinal detachment after Nd:YAG capsulotomy for PCO was rare in eyes that had previous uneventful phacoemulsification and PC IOL implantation. The exact relationship between the 2 events remains to be established.

Purpose: To determine whether uneventful extracapsular cataract extraction (ECCE) with posterior chamber intraocular lens (PC IOL) implantation induces epiretinal membranes (ERMs)., Setting: Private practice, with statistical evaluation at a university department in Germany., Methods: This prospective study comprised 296 consecutive patients (332 eyes) who had uneventful ECCE with PC IOL implantation for senile cataract. Eyes were examined within 2 weeks preoperatively and 2 weeks and 6, 9, and 12 months postoperatively. The macula was examined with a 78.0 diopter Volk lens. The main outcome measure was the presence of ERM. The membranes were classified as present when cellophane macular reflex (CMR) or macular pucker (MP) was observed., Results: The median age of the patients was 78 years (range 50 to 97 years). At baseline, ERM was present in 49 of 332 eyes (14.8%), 40 (12.1%) with CMR and 9 (2.7%) with MP. Six months postoperatively, ERM was present in 84 of 332 eyes (25.3%), 72 (21.7%) with CMR and 12 (3.6%) with MP. The difference between the baseline and 6 month incidence was significant (P <.001). All new cases of ERM were of the CMR type. One year after surgery, ERM was present in 54 of 198 eyes (27.3%), 47 (23.7%) with CMR and 7 (3.5%) with MP. The ERMs rarely influenced visual acuity; however, 6 of 12 eyes (50.0%) with MP and 13 of 72 (18.1%) with CMR had metamorphopsia., Conclusions: The prevalence of ERM increased by 71.4% during the first 6 months after uneventful ECCE with PC IOL implantation. Thereafter, the prevalence remained about the same. Newly formed ERMs were probably induced by the uneventful surgery. Although new-onset membranes usually do not preclude good visual acuity, they can cause metamorphopsia postoperatively.

Background: Results from epidemiologic studies suggest that cataract extraction is a risk-factor for the formation of epiretinal membranes (EM)., Patients and Methods: 250 consecutive eyes which underwent uneventful phakoemulsification with implantation of a posterior chamber intraocular lens were examined within two weeks prior and an two weeks after surgery for the presence of epiretinal membranes. Cellophane macular reflex (CMR) was distinguished from macular pucker (MP)., Results: We found an increase in the prevalence of epiretinal membranes from preoperatively 12/250 (4.8%) to post-operatively 33/250 (13.2%). The prevalence of CMR increased from 8/250 (3.2%) to 27/250 (10.8%). The prevalence of MP remained almost the same 4/250 (1.6%) prior and 6/250 (2.4%) after surgery., Conclusions: Increased prevalence of epiretinal membranes following cataract extraction observed in epidemiological studies can be explained by easier detection of such membranes in pseudophakic eyes where the observer gets a clear view of the fundus. Therefore it is questionable whether uneventful extracapsular cataract extraction can induce the formation of epiretinal membranes.

Purpose: To ascertain whether phacoemulsification with posterior chamber intraocular lens (IOL) implantation causes long-term reduction in intraocular pressure (IOP)., Setting: Private practice, Kempten, Germany., Methods: Intraocular pressure was measured in both eyes of 120 consecutive patients who were unilaterally phakic after phacoemulsification a mean of 17 months +/- 17 (SD) previously. Mean age of the 36 men and 84 women was 76 +/- 10 years. Data were analyzed using binomial distribution and the Wilcoxon signed-rank test., Results: The median ratio of IOP in the pseudophakic eye to IOP in the phakic eye was 0.83. The IOP was lower in the pseudophakic eye in 96 patients (80%). The median IOP was 12 mm Hg in the pseudophakic eyes and 14 mm Hg in the phakic eyes (P < .001). As measured by the interquartile range, IOP distribution was more centered in the pseudophakic than in the phakic eyes (3 versus 4). The IOP in the pseudophakic eyes remained lower to the last measurement, 5 years postoperatively, and appeared to be independent of patient age. Lower IOP in the pseudophakic eye was consistently present in patients with higher IOP in the phakic eye (16 to 22 mm Hg)., Conclusion: Phacoemulsification with posterior chamber IOL implantation reduced IOP in most but not all patients with a preoperative IOP of 22 mm Hg or less. This reduction was maintained over several years, with the cause yet to be established. Lower IOP may decrease the risk of subsequent glaucomatous nerve damage in these patients.

Uncomplicated extracapsular cataract extraction (EC) followed by posterior chamber lens implantation (PCL) has been shown to cause long-term lowering of intraocular pressure (IOP) in most patients. Since it has been suggested that Neodymium: YAG (Nd:YAG) laser capsulotomy can provoke persistent glaucoma, we examined the hypothesis that Nd:YAG laser capsulotomy may lead to a permanent elevation of IOP. In a case-control study, we compared two groups of 75 normotensive patients, who were unilaterally pseudophakic after phacoemulsification (PE) + PCL. Patients in group 1 had undergone Nd:YAG capsulotomy at least 2 months prior to this study, while in group 2 the posterior capsule was intact. Both groups were selected at random. Prior to surgery, none of the patients had suffered from glaucoma. Goldmann applanation tonometry was performed in both eyes of each patient and from that the ratio of IOP(pseudophakic)/IOP(phakic) was calculated. The distribution of this ratio for the two groups was compared using the Wilcoxon signed-rank test. There were 52 female and 23 male patients in group 1 versus 56 female and 19 male patients in group 2. Their mean ages were 75 + or -11 (group 1) and 75 + or - 9 years (group 2). The mean interval after PE + PCL was 48 + or - 29 months for patients with Nd:YAG capsulotomy and 16 + or - 17 months for patients with intact posterior capsule. Since the Nd:YAG capsulotomy, 28 + or - 23 months had elapsed on average. The median ratio of IOP (pseudophakic)/IOP(phakic) was higher in the Nd:YAG capsulotomy group than in the group of patients with an intact posterior capsule (1.00 vs. 0.80; p < 0.0001). There was almost no percentile, for which the distribution curve of group 1 intersected the curve of group 2. The median IOP in pseudophakic eyes was 14 mm Hg in group 1 and 12 mm Hg in group 2 (p<0.0001). These results are in accordance with the hypothesis that Nd:YAG capsulotomy will raise IOP permanently in most patients. Therefore Nd:YAG capsulotomy may harbor the risk of glaucomatous optic nerve damage in the long run. Long-term follow-up seems advisable in order to prevent possible glaucoma damage.

Background: Extracapsular cataract extraction with implantation of a posterior chamber lens (ECCE+PCL) lowers intraocular pressure (IOP) in most eyes. However it is believed that this effect vanishes with time., Patients and Methods: We measured IOP by Goldmann applanation tonometry in both eyes of 100 consecutive patients who were either bilaterally phakic, unilaterally pseudophakic or bilaterally pseudophakic. The posterior capsule was intact in all pseudophakic eyes and none of the eyes suffered from glaucoma., Results: Median IOP was 12 mmHg for pseudophakic and 14 mm Hg for phacic eyes (p < 0.0001). The median ratio of IOPpseudophakic/IOPphakic (0.82) in unilaterally pseudophakics was significantly lower than the median ratio of IOP1. pseudophakic eye/IOP2. pseudophakic eye (1.02) in bilaterally pseudophakics (p < 0.0001). The ratio in bilaterally pseudophakic subjects was independent of the time passed since ECCE+PCL. The distribution of the ratio of IOPs was similar for the group of bilaterally pseudophakics and bilaterally phakics., Conclusion: We conclude that the amount of lowering of IOP induced by ECCE+PCL is characteristic for each patient. It is well reproducible, when the second eye is operated. The lowering of IOP does not fade with time but is stable. Possibly ECCE+PCL lowers the risk to develop glaucomatous optic nerve damage permanently.

It is suspected that PE+PCL will often lower IOP. IOP was measured by Goldmann applanation tonometry in 50 consecutive patients (12 males and 38 females) who were unliterally pseudophacic (28 right and 22 left eyes n.s.). They had an intact posterior capsule and were not suffering from glaucoma. Mean age was 76 +/- 10 years. Time intervals since cataract surgery ranged from 2 to 68 months (mean 15 +/- 17 months). Seventy-four percent (99% confidence interval: 61% < P < 87%) of the patients showed lower IOP (less than 90% of reference value) in the pseudophacic eye. Median IOP in pseudophacic eye was 12 mmHg vs 14 mmHg in phacic control eyes (P < 0.01). These results are in accordance with the hypothesis that PE+PCL usually leads to a long-term reduction in IOP.

Apolipoprotein E isoforms were determined in 139 unrelated patients with retinitis pigmentosa (RP). When compared to prevalence rates for the general population in Germany, an increased prevalence was observed for phenotypes E2/E2: 10.1 vs. 1.0% (p less than 0.001), E2/E3: 19.4 vs. 12.0% (p less than 0.05), and E2/E4: 5.8 vs. 1.5% (n.s.), while the prevalence appeared to be reduced for phenotypes E3/E3: 48.9 vs. 59.8% (n.s.) E3/E4: 13.7 vs. 22.9% (p less than 0.05), and E4/E4: 2.2 vs. 2.8% (n.s.). These findings suggest that genetically determined abnormalities of plasma lipoprotein metabolism may be associated with some forms of RP.

The lipid composition of human aqueous humor was determined in ten samples from patients undergoing cataract surgery. The mean concentration was 16.4 mg/dl for total lipids, 2.5 for phospholipids, 1.1 for free fatty acids, 1.7 for unesterified cholesterol 2.0 for triglycerides, and 9.0 mg/dl for cholesterol esters, respectively. The presence of lipids in aqueous humor suggests that lipids are transported through the human aqueous humor.

The nature and etiology of plasma lipoprotein abnormalities in patients with varying stages of primary biliary cirrhosis (PBC) were assessed. Two distinct lipoprotein patterns were observed. In patients with early and intermediate histologic stages of PBC, mild elevations of very low density lipoproteins and low density lipoproteins (LDLs), and marked increases in high density lipoproteins (HDLs) were often noted (group 1). In contrast, patients with advanced disease had marked elevations in LDLs with the presence of lipoprotein-X, and a significant decrease in HDLs (group 2). The lipoprotein pattern in group 1 was characterized by a normal ratio of free cholesterol to total cholesterol, whereas in group 2, this ratio was elevated. In plasma from group 1 patients only spherical micelles were observed on electron microscopy, whereas in plasma from group 2 patients bilayered disks could be seen in LDLs and HDLs. The increase in HDLs observed in group 1 subjects was associated with elevations in HDL2, whereas in group 2 subjects only very low amounts of HDL3 were observed on analytic ultracentrifugation. Mean plasma apolipoprotein B and C-II concentrations were increased in both groups; but apolipoprotein A-I and AII values were divergent, with group 1 subjects having elevated values,and group 2 subjects having decreases values. Mean postheparin hepatic lipase activity was decreased in both groups of patients with PBC due to the presence of an inhibitor in PBC plasma, while altered cholesterol esterification was only observed in group 2. These data indicate that hepatic lipase inhibition may play an important role in the pathogenesis of the lipoprotein abnormalities seen in early and intermediate PBC, and these abnormalities are then further modified by altered cholesterol esterification in advanced disease.

To determine the possible role of glucose and lipid metabolism in the formation of cataract in elderly people we studied 463 patients undergoing cataract extraction. Of 188 males, 35 (19%) had posterior subcapsular cataract (group 1), 27 (14%) had cortical cataract (group 2), and 24 (13%) had nuclear cataract (group 3). Of 275 females, 27 (10%) belonged to group 1, 44 (16%) to group 2, and 33 (12%) to group 3. Patients in group 1 were significantly younger than those of group 2 and 3. In addition, patients in group 1 had higher concentrations of fasting serum triglycerides than patients of group 2 or 3. No difference in mean concentrations of serum cholesterol was observed between the different groups. However, fasting plasma glucose concentrations were higher in group 1 patients than in those of group 2 or 3. Patients in group 1 had a higher 'Broca index' than those in group 2 or 3, with the exception of males in group 3. These results suggest that the association of hypertriglyceridemia, hyperglycemia and obesity favors the formation of a specific morphologic type of lens opacity, posterior subcapsular cataract, occurring at an early age. They imply the possibility of effective modes of preventive therapy for a subgroup of patients with 'senile' cataract.