Comparison of microbial community assemblages in the rhizosphere of three Amaranthus spp.

Weed management remains a major challenge in cropping systems worldwide, with rising interest in ecological based approaches that can be integrated with herbicide use. Soil microbial communities may play important, yet undiscovered, roles in weed success. Little is known about the rhizosphere communities associated with weeds like Amaranthus, commonly known as pigweeds, and considered some of the most problematic weeds in agricultural systems. In a greenhouse experiment that allowed controlled plant growth conditions and a high number of individual plant specimens to analyze for statistical robustness (n = 8 per species), we show that specific bacterial assemblages form in the rhizospheres of A. retroflexus L. (redroot pigweed), A. palmeri S. Watson (Palmer amaranth), and A. tuberculatus (Moq.) J. D. Sauer (waterhemp). Using a relatively rapid and easy approach of T-RFLP community profiling of the 16S rRNA genes, distinct assemblages corresponded to plant species (PERMANOVA F = 14.776, p = 0.001), and further within each species, similar communities (F = 11.449, p = 0.001) were associated with three rhizosphere soil fractions taken in increasing distances away from the root tissue. These results provide the first solid basis for distinct plant-microbe relationships within three closely related Amaranthus species, warranting closer examination of the identities and function of the microorganisms that appear to be selectively recruited from the extant soil community. More intensive efforts to obtain the microbial taxonomic identities via sequencing are underway that can lead to further detailed studies to elucidate important functional plant-microbe interactions that may associate with weed success. Such data provides underlying key information that may ultimately exploit weed-microbe interactions in development of new integrated weed control tactics.

Tropical lacustrine sediment microbial community response to an extreme El Niño event.

Salinity can influence microbial communities and related functional groups in lacustrine sediments, but few studies have examined temporal variability in salinity and associated changes in lacustrine microbial communities and functional groups. To better understand how microbial communities and functional groups respond to salinity, we examined geochemistry and functional gene amplicon sequence data collected from 13 lakes located in Kiritimati, Republic of Kiribati (2° N, 157° W) in July 2014 and June 2019, dates which bracket the very large El Niño event of 2015-2016 and a period of extremely high precipitation rates. Lake water salinity values in 2019 were significantly reduced and covaried with ecological distances between microbial samples. Specifically, phylum- and family-level results indicate that more halophilic microorganisms occurred in 2014 samples, whereas more mesohaline, marine, or halotolerant microorganisms were detected in 2019 samples. Functional Annotation of Prokaryotic Taxa (FAPROTAX) and functional gene results (nifH, nrfA, aprA) suggest that salinity influences the relative abundance of key functional groups (chemoheterotrophs, phototrophs, nitrogen fixers, denitrifiers, sulfate reducers), as well as the microbial diversity within functional groups. Accordingly, we conclude that microbial community and functional gene groups in the lacustrine sediments of Kiritimati show dynamic changes and adaptations to the fluctuations in salinity driven by the El Niño-Southern Oscillation.

Stable carbon isotope values of syndepositional carbonate spherules and micrite record spatial and temporal changes in photosynthesis intensity.

Marine and lacustrine carbonate minerals preserve carbon cycle information, and their stable carbon isotope values (δ13 C) are frequently used to infer and reconstruct paleoenvironmental changes. However, multiple processes can influence the δ13 C values of bulk carbonates, confounding the interpretation of these values in terms of conditions at the time of mineral precipitation. Co-existing carbonate forms may represent different environmental conditions, yet few studies have analyzed δ13 C values of syndepositional carbonate grains of varying morphologies to investigate their origins. Here, we combine stable isotope analyses, metagenomics, and geochemical modeling to interpret δ13 C values of syndepositional carbonate spherules (>500 µm) and fine-grained micrite (<63 µm) from a ~1600-year-long sediment record of a hypersaline lake located on the coral atoll of Kiritimati, Republic of Kiribati (1.9°N, 157.4°W). Petrographic, mineralogic, and stable isotope results suggest that both carbonate fractions precipitate in situ with minor diagenetic alterations. The δ13 C values of spherules are high compared to the syndepositional micrite and cannot be explained by mineral differences or external perturbations, suggesting a role for local biological processes. We use geochemical modeling to test the hypothesis that the spherules form in the surface microbial mat during peak diurnal photosynthesis when the δ13 C value of dissolved inorganic carbon is elevated. In contrast, we hypothesize that the micrite may precipitate more continuously in the water as well as in sub-surface, heterotrophic layers of the microbial mat. Both metagenome and geochemical model results support a critical role for photosynthesis in influencing carbonate δ13 C values. The down-core spherule-micrite offset in δ13 C values also aligns with total organic carbon values, suggesting that the difference in the δ13 C values of spherules and micrite may be a more robust, inorganic indicator of variability in productivity and local biological processes through time than the δ13 C values of individual carbonate forms.

Differential structure and functional gene response to geochemistry associated with the suspended and attached shallow aquifer microbiomes from the Illinois Basin, IL.

Despite the clear ecological significance of the microbiomes inhabiting groundwater and connected ecosystems, our current understanding of their habitats, functionality, and the ecological processes controlling their assembly have been limited. In this study, an efficient pipeline combining geochemistry, high-throughput FluidigmTM functional gene amplification and sequencing was developed to analyze the suspended and attached microbial communities inhabiting five groundwater monitoring wells in the Illinois Basin, USA. The dominant taxa in the suspended and the attached microbial communities exhibited significantly different spatial and temporal changes in both alpha- and beta-diversity. Further analyses of representative functional genes affiliated with N2 fixation (nifH), methane oxidation (pmoA), and sulfate reduction (dsrB, and aprA), suggested functional redundancy within the shallow aquifer microbiomes. While more diversified functional gene taxa were observed for the suspended microbial communities than the attached ones except for pmoA, different levels of changes over time and space were observed between these functional genes. Notably, deterministic and stochastic ecological processes shaped the assembly of microbial communities and functional gene reservoirs differently. While homogenous selection was the prevailing process controlling assembly of microbial communities, the neutral processes (e.g., dispersal limitation, drift and others) were more important for the functional genes. The results suggest complex and changing shallow aquifer microbiomes, whose functionality and assembly vary even between the spatially proximate habitats and fractions. This research underscored the importance to include all the interface components for a more holistic understanding of the biogeochemical processes in aquifer ecosystems, which is also instructive for practical applications.

Hierarchical detection of diverse Clade II (atypical) nosZ genes using new primer sets for classical- and multiplex PCR array applications.

The reduction of nitrous oxide (N2O) to N2 represents the key terminal step in canonical denitrification. Nitrous oxide reductase (NosZ), the enzyme associated with this biological step, however, is not always affiliated with denitrifying microorganisms. Such organisms were shown recently to possess a Clade II (atypical) nosZ gene, in contrast to Clade I (typical) nosZ harbored in more commonly studied denitrifiers. Subsequent phylogenetic analyses have shown that Clade II NosZ are affiliated with a much broader diversity of microorganisms than those with Clade I NosZ, the former including both non-denitrifiers and denitrifiers. Most studies attempting to characterize the nosZ gene diversity using DNA-based PCR approaches have only focused on Clade I nosZ, despite recent metagenomic sequencing studies that have demonstrated the dominance of Clade II nosZ genes in many ecosystems, particularly soil. As a result, these studies have greatly underestimated the genetic potential for N2O reduction present in ecosystems. Because the high diversity of Clade II NosZ makes it impossible to design a universal primer set that would effectively amplify all nosZ genes in this clade, we developed a suite of primer sets to specifically target seven of ten designated subclades of Clade II nosZ genes. The new primer sets yield suitable product sizes for paired end amplicon sequencing and qPCR, demonstrated here in their use for both conventional single-reaction and multiplex array platforms. In addition, we show the utility of these primers for detecting nosZ gene transcripts from mRNA extracted from soil.

Sequence alignments and validation of PCR primers used to detect phylogenetically diverse nrfA genes associated with dissimilatory nitrate reduction to ammonium (DNRA).

PCR primer sets were designed to target nrfA, the gene encoding the pentaheme nitrite reductase NrfA that catalyzes the nitrite ammonification step in the process of dissimilatory nitrate reduction to ammonium (DNRA). Details of the nucleotide alignments of the primer target regions of 271 nrfA sequences from reference genomes representing 18 distinct clades of NrfA are shown here along with validation of application to PCR-based methodology including the use of amplified fragment length polymorphism (AFLP) profiling and Illumina platform amplicon-based sequencing of environmental samples and selected reference strains. Summary data tables illustrate the specificity of forward primers nrfAF2awMOD and nrfAF2awMODgeo when paired with the new reverse primer nrfAR1MOD in relation to consensus target reference sequences associated with members of 18 NrfA clades. Specificity of the new primers to nrfA sequences in environmental samples is shown in AFLP analysis and amino acid-translated amplicon sequences obtained with the new primer sets. We also provide sequence alignment files of the full length nrfA genes, PCR reference amplicon alignment, NrfA amino-acid alignment and NrfA translated PCR amplicon-amino acid alignment. The full nucleotide and protein alignments contain 271 reference genomes that represent the 18 identified NrfA clades as a tool to further aid practitioners in examining new sequences corresponding to the primer target regions and allow further primer design modifications if deemed pertinent to specific studies. A more comprehensive analysis of this data may be obtained from ("Optimization of PCR primers to detect phylogenetically diverse nrfA genes associated with nitrite ammonification" Cannon et al., 2019).

Optimization of PCR primers to detect phylogenetically diverse nrfA genes associated with nitrite ammonification.

Dissimilatory nitrate reduction to ammonium (DNRA) is now known to be a more prevalent process in terrestrial ecosystems than previously thought. The key enzyme, a pentaheme cytochrome c nitrite reductase NrfA associated with respiratory nitrite ammonification, is encoded by the nrfA gene in a broad phylogeny of bacteria. The lack of reliable and comprehensive molecular tools to detect diverse nrfA from environmental samples has hampered efforts to meaningfully characterize the genetic potential for DNRA in environmental systems. In this study, modifications were made to optimize the amplification efficiency of previously-designed PCR primers, targeting the diagnostic region of NrfA between the conserved third- and fourth heme binding domains, and to increase coverage to include detection of environmentally relevant Geobacteraceae-like nrfA. Using an alignment of the primers to >270 bacterial nrfA genes affiliated with 18 distinct clades, modifications to the primer sequences improved coverage, minimized amplification artifacts, and yielded the predicted product sizes from reference-, soil-, and groundwater DNA. Illumina sequencing of amplicons showed the successful recovery of nrfA gene fragments from environmental DNA based on alignments of the translated sequences. The new primers developed in this study are more efficient in PCR reactions, although gene targets with high GC content affect efficiency. Furthermore, the primers have a broader spectrum of detection and were validated rigorously for use in detecting nrfA from natural environments. These are suitable for conventional PCR, qPCR, and use in PCR access array technologies that allow multiplex gene amplification for downstream high throughput sequencing platforms.

Identification of metolachlor mineralizing bacteria in aerobic and anaerobic soils using DNA-stable isotope probing.

The influence of soil environmental factors such as aeration on the ecology of microorganisms involved in the mineralization and degradation of the popular soil-applied pre-emergent herbicide, metolachlor is unknown. To address this knowledge gap, we utilized DNA-based stable isotope probing (SIP) where soil microcosms were incubated aerobically or anaerobically and received herbicide treatments with unlabeled metolachlor or 13C-metolachlor. Mineralization of metolachlor was confirmed as noted from the evolution of 14CO2 from 14C-metolachlor-treated microcosms and clearly demonstrated the efficient utilization of the herbicide as a carbon source. Terminal restriction fragment length polymorphisms (T-RFLP) bacterial community profiling performed on soil DNA extracts indicated that fragment 307 bp from aerobic soil and 212 bp from anaerobic soil were detected only in the herbicide-treated (both unlabeled metolachlor and 13C-metolachlor) soils when compared to the untreated control microcosms. T-RFLP profiles from the ultracentrifugation fractions illustrated that these individual fragments experienced an increase in relative abundance at a higher buoyant density (BD) in the labeled fractions when compared to the unlabeled herbicide amendment fractions. The shift in BD of individual T-RFLP fragments in the density-resolved fractions suggested the incorporation of 13C from labeled herbicide into the bacterial DNA and enabled the identification of organisms responsible for metolachlor uptake from the soil. Subsequent cloning and 16S rRNA gene sequencing of the 13C-enriched fractions implicated the role of organisms closely related to Bacillus spp. in aerobic mineralization and members of Acidobacteria phylum in anaerobic mineralization of metolachlor in soil.

Refined NrfA phylogeny improves PCR-based nrfA gene detection.

Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are contrasting microbial processes in the terrestrial nitrogen (N) cycle, in that the former promotes N retention and the latter leads to N loss (i.e., the formation of gaseous products). The nitrite reductase NrfA catalyzes nitrite reduction to ammonium, the enzyme associated with respiratory nitrite ammonification and the key step in DNRA. Although well studied biochemically, the diversity and phylogeny of this enzyme had not been rigorously analyzed. A phylogenetic analysis of 272 full-length NrfA protein sequences distinguished 18 NrfA clades with robust statistical support (>90% Bayesian posterior probabilities). Three clades possessed a CXXCH motif in the first heme-binding domain, whereas all other clades had a CXXCK motif in this location. The analysis further identified a KXRH or KXQH motif between the third and fourth heme-binding motifs as a conserved and diagnostic feature of all pentaheme NrfA proteins. PCR primers targeting a portion of the heme-binding motifs that flank this diagnostic region yielded the expected 250-bp-long amplicons with template DNA from eight pure cultures and 16 new nrfA-containing isolates. nrfA amplicons obtained with template DNA from two geomorphically distinct agricultural soils could be assigned to one of the 18 NrfA clades, providing support for this expanded classification. The extended NrfA phylogeny revealed novel diagnostic features of DNRA populations and will be useful to assess nitrate/nitrite fate in natural and engineered ecosystems.

Bisphenol A-mediated suppression of LPL gene expression inhibits triglyceride accumulation during adipogenic differentiation of human adult stem cells.

The endocrine disrupting chemical, bisphenol A (BPA), has been shown to accelerate the rate of adipogenesis and increase the amount of triglyceride accumulation during differentiation of 3T3-L1 preadipocytes. The objective of this study was to investigate if that observation is mirrored in human primary cells. Here we investigated the effect of BPA on adipogenesis in cultured human primary adult stem cells. Continuous exposure to BPA throughout the 14 days of differentiation dramatically reduced triglyceride accumulation and suppressed gene transcription of the lipogenic enzyme, lipoprotein lipase (LPL). Results presented in the present study show for the first time that BPA can reduce triglyceride accumulation during adipogenesis by attenuating the expression of LPL gene transcription. Also, by employing image cytometric analysis rather than conventional Oil red O staining techniques we show that BPA regulates triglyceride accumulation in a manner which does not appear to effect adipogenesis per se.

Structural characterization of Haemophilus parainfluenzae lipooligosaccharide and elucidation of its role in adherence using an outer core mutant.

The opportunistic pathogen Haemophilus parainfluenzae is a gram-negative bacterium found in the oropharynx of humans. Haemophilus parainfluenzae is a member of the Pasteurellaceae family in which it is most closely related to Haemophilus sengis and Actinobacillus. Characterization of surface displayed lipooligosaccharide has identified components that are crucial in adherence. We examined the oligosaccharide structure of lipooligosaccharide from 2 clinical isolates of H. parainfluenzae. Core oligosaccharide was isolated by standard methods from purified lipooligosaccharide. Structural information was established by a combination of monosaccharide and methylation analyses, nuclear magnetic resonance spectroscopy, and mass spectrometry revealing the following structures: R-(1-6)-beta-Glc-(1-4)-D,D-alpha-Hep-(1-6)-beta-Glc-(1-4)- substituting a tri-heptose-Kdo inner core of L,D-alpha-Hep-(1-2)-L,D-alpha-Hep-(1-3)-L,D-alpha-Hep-(1-5)-alpha-Kdo at the 4-position of the proximal L,D-alpha-Hep residue to Kdo, and with a PEtn residue at the 6-position of the central L,D-alpha-Hep residue. In strain 4282, the R substituent is beta-galactose and in strain 4201 there is no substituent at the distal glucose. These analyses have revealed that multiple structural aspects of H. parainfluenzae lipooligosaccharide are comparable with nontypeable Haemophilus influenzae lipooligosaccharide. This study also identified a galactan in strain 4201 and a glucan in strain 4282. Haemophilus parainfluenzae was shown to adhere to a bronchial epithelial cell line to the same degree as nontypeable H. influenzae. However, an H. parainfluenzae mutant lacking the outer core of the lipooligosaccharide showed diminished adherence to the epithelial cells, suggesting that H. parainfluenzae lipooligosaccharide plays a role in tissue colonization.

Efficacy of anal fistula plug in closure of cryptoglandular fistulas: long-term follow-up.

PURPOSE: The long-term efficacy of Surgisis anal fistula plug in closure of cryptoglandular anorectal fistulas was studied. METHODS: Patients with high cryptoglandular anorectal fistulas were prospectively studied. Additional variables recorded were: number of fistula tracts, and presence of setons. Under general anesthesia and in prone jackknife position, patients underwent irrigation of the fistula tract by using hydrogen peroxide. Each primary opening was occluded by using a Surgisis anal fistula plug, which was securely sutured in place at the primary opening and tacked to the periphery of the secondary opening. RESULTS: Forty-six patients were prospectively enrolled during a two-year period. Follow-up was six months to two years (median, 12 months). At final follow-up, all fistula tracts had been successfully closed in 38 patients, for an overall success rate of 83 percent. Seven patients had multiple tracts, for a total of 55 fistula tracts in the series. Of the 55 individual tracts, 47 (85 percent) were closed at final follow-up. Patients with one primary opening were most likely to have successful closure by using the anal fistula plug, although this was not significant. Successful closure was not correlated with the presence of setons. CONCLUSIONS: Long-term closure of cryptoglandular anorectal fistula tracts using Surgisis anal fistula plug is safe and successful in 83 percent of patients and 85 percent of tracts.

Efficacy of anal fistula plug in closure of Crohn's anorectal fistulas.

PURPOSE: The efficacy of Surgisis anal fistula plug in closure of Crohn's anorectal fistula was studied. METHODS: Patients with Crohn's anorectal fistulas were prospectively studied. Diagnosis was made by histologic, radiographic, or endoscopic criteria. Variables recorded were: number of fistula tracts (primary openings), presence of setons, and current antitumor necrosis factor therapy. Under general anesthesia and in prone jackknife position, patients underwent irrigation of the fistula tract by using hydrogen peroxide. Each primary opening was occluded by using a Surgisis anal fistula plug. Superficial tracts amenable to fistulotomy were excluded. RESULTS: Twenty consecutive patients were prospectively enrolled, comprising a total of 36 fistula tracts. At final follow-up, all fistula tracts had been successfully closed in 16 of 20 patients, for an overall success rate of 80 percent. Thirty of 36 individual fistula tracts (83 percent) were closed at final follow-up. Patients with single fistulas (with 1 primary opening) were most likely to have successful closure using the anal fistula plug. Successful closure was not correlated with the presence of setons or antitumor necrosis factor therapy. CONCLUSIONS: Closure of Crohn's anorectal fistula tracts using Surgisis anal fistula plug is safe and successful in 80 percent of patients and 83 percent of fistula tracts. Closure rates were higher with single tracts than complex fistulas with multiple primary openings.

Forgivingness, vengeful rumination, and affective traits.

Trait forgivingness is the disposition to forgive interpersonal transgressions over time and across situations. We define forgiveness as the replacement of negative unforgiving emotions with positive, other-oriented emotions. Rumination has been suggested as a mediator between forgivingness and emotional outcomes; however, we suggest that different content of rumination leads to different outcomes after transgressions. In four studies of 179, 233, 80, and 66 undergraduate students, trait forgivingness was negatively correlated with trait anger, hostility, neuroticism, fear, and vengeful rumination and was positively correlated with agreeableness, extraversion, and trait empathy. The disposition to ruminate vengefully mediated the relationship between trait forgivingness and (1) anger-related traits and (2) both revenge motivations and state anger following a specific recent transgression, but it did not mediate between forgivingness and (1) fearfulness and (2) avoidance motivations following a specific transgression. Self-hate statements, a proxy for depressive rumination, mediated the relationship between forgivingness and both depression and fearfulness but not the relationship between forgivingness and trait anger. Future research should distinguish the contents of mental rumination following interpersonal transgressions.

Guilt, fear, submission, and empathy in depression.

BACKGROUND: This study compares self-focused motivations (fear of negative evaluation, social comparison, and fear of envy) and other-focused motivations (empathy and interpersonal guilt) in submissive behavior and depression. METHODS: The Beck Depression Inventory, Submissive Behavior Scale, Fear of Negative Evaluation Scale, Social Comparison Scale, Interpersonal Guilt Questionnaire, and Interpersonal Reactivity Inventory were administered to 50 patients hospitalized for depression and 52 students. RESULTS: Depressed patients were significantly higher in survivor guilt, omnipotent responsibility guilt, submissive behavior, fear of negative evaluation, fear of envy, and empathic distress, and lower in social comparison. LIMITATIONS: This research was limited in that it was a correlational study. CONCLUSIONS: This study suggests that altruistic concern about others may be an important factor in depression and submissive behavior. Evolutionary implications of these findings are discussed.

[The German short version of the interpersonal guilt questionnaire--Validation in a population-based sample and clinical application]. / Der "Fragebogen zu interpersonellen Schuldgefühlen" (FIS) - Anwendung in einer repräsentativen Bevölkerungsstichprobe und bei PsychotherapiepatientInnen.

The present study describes the development of the German short version "Fragebogen zu interpersonellen Schuldgefühlen" (FIS) of the "Interpersonal Guilt Questionnaire" (IGQ) developed by O'Connor et al. The questionnaire was used in a representative sample of 652 eastern and 1283 western Germans. Woman show higher scores on the subscales "Seperation Guilt" and "Omnipotent Responsibility Guilt" than man. Eastern Germans mark higher scores on the subscale "Omnipotent Responsibility Guilt" than western Germans. Correlations between interpersonal guilt and social anxiety and differences between a non-clinical and a clinical sample show that maladaptive aspects of guilt feelings can be assessed by the "Interpersonal Guilt Questionnaire".

Role of steroids in sex differences in morphine-induced analgesia: activational and organizational effects.

The purpose of the present studies was to determine the role of either the organizational or activational sex steroids in mediating the sex differences observed in morphine-induced antinociception in the rat. To examine the organizational aspects, male pups were castrated at postnatal days 1 and 2; females were masculinized by large doses of testosterone on postnatal days 1 and 2. Adult male and female rats were also castrated over a period of 2 months to examine the role of the acute activational effects of the opiates in the already sexually differentiated adult rat brain. The results of these studies demonstrate that there were no alterations in the sex differences in opiate analgesia in castrated adult male and female rats; thus, male- and female-specific responses to opiate-induced antinociception were maintained even in the absence of the acute membrane-mediated effects of sex steroids. On the other hand, in male rats, castrated at postnatal days 1 and 2, the morphine dose-response curve shifted markedly to the right and, in fact, was almost identical to that observed in untreated females. Conversely, in female rats, masculinized by large doses of testosterone early in prenatal life, the morphine dose-response curve shifted to the left, yielding a dose-response curve that resembled that in normal males. These results strongly suggest that the sex differences that have been observed in morphine-induced analgesia are due to the organizational effects of sex steroids in the developing rat brain, rather than their acute activational effects in adulthood.

Gene regulation by low level UV-B radiation: identification by DNA array analysis.

UV-B radiation alters transcript levels of various defence genes and photosynthetic genes in plants. Utilising a DNA array with 5000 ESTs and cDNAs from Arabidopsis thaliana, 70 genes were found to show a greater than two-fold induction or repression of transcript levels. Six genes (MEB5.2, PyroA, Ubq3, Lhcb6, F5D21.10 and the gene for an RNA polymerase II subunit) were tested for stress specific gene regulation on northern blots with RNA from plants exposed to low dose UV-B radiation, ozone or wounding. Transcript levels for PyroA, Uhq3 and the gene for a RNA polymerase II subunit were all specifically increased by UV-B. MEB5.2 mRNA levels also rose, whereas Lhcb6 and FSD21.10 transcript levels decreased under all stresses. The PyroA gene product in fungi is needed for biosynthesis of pyridoxine, and might have a role in protection against singlet oxygen. The Ubq3 gene encodes the ubiquitin protein that is attached to proteins destined for degradation. MEB5.2 and F5D21.10 represent novel gene products whose function have not yet been identified. Pairwise comparisons between the UV-B inducible promoters have identified a series of elements present in the MEB5.2 and PyroA promoters, absent from promoters of genes for early phenylpropanoid metabolism and that may be responsible for modulating their UV-B responses.