Descriptive analyses of bacterial communities in marine sediment microcosms spiked with fish wastes, emamectin benzoate, and oxytetracycline.

In marine sediments surrounding salmon aquaculture sites, organic matter (OM) enrichment has been shown to influence resident bacterial community composition; however, additional effects on these communities due to combined use of the sea-lice therapeutant emamectin benzoate (EMB) and the widely used antibiotic oxytetracycline (OTC) are unknown. Here, we use sediment microcosms to assess the influence of OM, EMB, and OTC on benthic bacterial communities. Microcosms consisted of mud or sand sediments enriched with OM (fish and feed wastes) and spiked with EMB and OTC at environmentally-relevant concentrations. Samples were collected from initial matrices at the initiation of the trial and after 110 days for 16 S rRNA gene sequencing of the V3-V4 region and microbiome profiling. The addition of OM in both mud and sand sediments reduced alpha diversities; for example, an average of 1106 amplicon sequence variants (ASVs) were detected in mud with no OM addition, while only 729 and 596 ASVs were detected in mud with low OM and high OM, respectively. Sediments enriched with OM had higher relative abundances of Spirochaetota, Firmicutes, and Bacteroidota. For instance, Spirochaetota were detected in sediments with no OM with a relative abundance range of 0.01-1.2%, while in sediments enriched with OM relative abundance varied from 0.16% to 26.1%. In contrast, the addition of EMB (60 ng/g) or OTC (150 ng/g) did not result in distinct taxonomic shifts in the bacterial communities compared to un-spiked sediments during the timeline of this experiment. EMB and OTC concentrations may have been below effective inhibitor concentrations for taxa in these communities; further work should explore gene content and the presence of antibiotic resistance genes (ARGs) in sediment-dwelling bacteria.

Impact of Grandchild Caregiving on African American Grandparents.

The aim of this study was to describe the context and impact of caregiving for grandchildren with health concerns on grandparents. The study sample comprised 391 African American grandparents aged 55 or older. Logistic regression analysis indicated that grandparent caregivers of grandchildren with psychiatric or behavioral problems were more likely to experience a negative impact on their health (AOR = 7.86, p =.008) and leisure (AOR = 14.31, p =.024) than grandparent caregivers of grandchildren with no or other types of health problems. The findings underscore the need to support African American grandparent caregivers, particularly those raising grandchildren with mental health problems.

Cloacimonadota metabolisms include adaptations in engineered environments that are reflected in the evolutionary history of the phylum.

Phylum Cloacimonadota (previously Cloacimonetes, WWE1) is an understudied bacterial lineage frequently associated with engineered and wastewater systems. Cloacimonadota members were abundant and diverse in metagenomic datasets from a municipal landfill, prompting an examination of phylogenetic relationships, metabolic diversity, and pangenomic dynamics across the phylum, based on the 30 publicly available genomes and 24 new metagenome-assembled genomes (MAGs) from landfill samples. We found that Cloacimonadota have distinct evolutionary histories associated with engineered versus natural environments and identified genomic features and metabolic strategies that correlate to habitat of origin. Metabolic reconstructions for MAGs predict an anaerobic, acetogenic, and mixed fermentative and flavin-bifurcation-based anaerobic respiratory lifestyle for the majority of Cloacimonadota surveyed. Genomes from engineered ecosystems encode a suite of genes not typically found in genomes from natural environments including acetate kinase, genes for cysteine degradation to pyruvate, increased diversity of carbon utilization enzymes, and different mechanisms for generating membrane potential and ATP synthesis. This phylum-level examination also clarifies the distribution of functions previously observed for members of the phylum, where propionate oxidation and reverse TCA cycles are not common components of Cloacimonadota metabolism.

Efficient nuclear export of herpes simplex virus 1 transcripts requires both RNA binding by ICP27 and ICP27 interaction with TAP/NXF1.

Herpes simplex virus 1 (HSV-1) regulatory protein ICP27 has been reported to bind viral RNA and to interact with the nuclear export adaptor Aly/REF and the major cellular mRNA nuclear export receptor TAP/NXF1. Using in situ hybridization and in vitro export assays, we show here that poly(A)(+) RNA was retained in the nucleus of cells infected with viral ICP27 mutants that either cannot bind RNA or that do not interact with TAP/NXF1. Microarray analysis of nuclear and cytoplasmic RNA fractions demonstrated that efficient export of the majority of viral transcripts requires that ICP27 be able to bind RNA and to interact with TAP/NXF1. We conclude that ICP27 is the major export adaptor for HSV-1 mRNA and that it links bound transcripts to the TAP/NXF1 export receptor.

The cellular RNA export receptor TAP/NXF1 is required for ICP27-mediated export of herpes simplex virus 1 RNA, but the TREX complex adaptor protein Aly/REF appears to be dispensable.

Herpes simplex virus 1 (HSV-1) protein ICP27 has been shown to shuttle between the nucleus and cytoplasm and to bind viral RNA during infection. ICP27 was found to interact with the cellular RNA export adaptor protein Aly/REF, which is part of the TREX complex, and to relocalize Aly/REF to viral replication sites. ICP27 is exported to the cytoplasm through the export receptor TAP/NXF1, and ICP27 must be able to interact with TAP/NXF1 for efficient export of HSV-1 early and late transcripts. We examined the dynamics of ICP27 movement and its localization with respect to Aly/REF and TAP/NXF1 in living cells during viral infection. Recombinant viruses with a yellow fluorescent protein (YFP) tag on the N or C terminus of ICP27 were constructed. While the N-terminally tagged ICP27 virus behaved like wild-type HSV-1, the C-terminally tagged virus was defective in viral replication and gene expression, and ICP27 was confined to the nucleus, suggesting that the C-terminal YFP tag interfered with ICP27's C-terminal interactions, including the interaction with TAP/NXF1. To assess the role of Aly/REF and TAP/NXF1 in viral RNA export, these factors were knocked down using small interfering RNA. Knockdown of Aly/REF had little effect on the export of ICP27 or poly(A)(+) RNA during infection. In contrast, a decrease in TAP/NXF1 levels severely impaired export of ICP27 and poly(A)(+) RNA. We conclude that TAP/NXF1 is essential for ICP27-mediated export of RNA during HSV-1 infection, whereas Aly/REF may be dispensable.

Distribution of reactive oxygen species defense mechanisms across domain bacteria.

Bacteria are the most diverse and numerous organisms on the planet, inhabiting environments from the deep subsurface to particles in clouds. Across this range of conditions, bacteria have evolved a diverse suite of enzymes to mitigate cellular damage from reactive oxygen species (ROS). Here, we review the diversity and distribution of ROS enzymatic defense mechanisms across the domain Bacteria, using both peer-reviewed literature and publicly available genome databases. We describe the specific strategies used by well-characterized organisms in order to highlight differences in oxidative stress responses between aerobic, facultatively anaerobic, and anaerobic lifestyles. We present evidence from genome minimization experiments to suggest that ROS defenses are obligately required for life. This review clarifies the variability in ROS defenses across Bacteria, including the novel diversity found in currently uncharacterized Candidate Phyla.

A directional recombination cloning system for restriction- and ligation-free construction of GFP, DsRed, and lacZ transgenic Drosophila reporters.

The fruit fly Drosophila is a leading model system for the study of transcriptional control by cis-regulatory elements, or enhancers. Here we present a rapid, high-efficiency system for directionally cloning PCR-amplified, PCR-mutated, or synthetic enhancer sequences into the Ganesh family of P element reporter constructs, which contain reporter genes encoding nuclear-localized eGFP, DsRed, or beta-galactosidase. This system, which is scalable for either small projects or high-throughput approaches, makes use of both TOPO and Gateway cloning technologies for directional, efficient cloning, without the need for restriction digestion or ligation reactions. It should be especially useful for those researchers who wish to test large numbers of putative enhancers, those who are undertaking detailed mutational analyses of enhancer sequences, or those who wish to avoid the difficulties sometimes encountered in traditional cloning strategies.

Characterization of Brachyspira communities from clinical cases of swine mucohaemorrhagic diarrhea through deep sequencing of the NADH oxidase (nox) gene.

Swine dysentery is traditionally associated with Brachyspira hyodysenteriae, but the re-emergence of Brachyspira-associated disease in North America associated with a novel causative species, B. hampsonii, is now a concern for swine producers. The pathogenesis of Brachyspira-associated disease is not completely understood, and it is not known whether mixed infections of Brachyspira spp. are important in disease development. Deep sequencing of partial sequences of the nox gene amplified with genus-specific primers was used to detect Brachyspira spp. in 55 fecal samples from clinical cases of mucohaemorrhagic diarrhea in pigs from Western Canada that had been identified as positive for one or more Brachyspira species using established diagnostic tests. Synthetic mixtures of Brachyspira genomic DNA were included in the study to define detection limits for the technique and identify biases in detection of different species. Multiple species were detected in all clinical cases for which sufficient nox sequence data were generated (n = 47), indicating that mixed species Brachyspira infections are common, although in most cases, one species accounted for at least half of the sequences identified. In all cases, the species detected in the original diagnostic investigation of each case was also detected by nox sequencing. Results from synthetic communities indicated that the method was highly reproducible, but also indicated potential PCR bias against B. hampsonii genomovar I. Deep sequencing of the nox gene target is a suitable method for simultaneous detection of multiple Brachyspira species in clinical case material that may offer advantages over current, more targeted diagnostic approaches for investigating the significance of mixed infections in disease development.

Optimizing a PCR protocol for cpn60-based microbiome profiling of samples variously contaminated with host genomic DNA.

BACKGROUND: The current recommended protocol for chaperonin-60 (cpn60) universal target based microbiome profiling includes universal PCR of microbiome samples across an annealing temperature gradient to maximize the diversity of sequences amplified. However, the value of including this gradient approach has not been formally evaluated since the optimization of a modified universal PCR primer cocktail for cpn60 PCR. PCR conditions that maximize representation of the microbiome while minimizing PCR-associated distortion of the community structure, especially in samples containing large amounts of host genomic DNA are critical. The goal of this study was to measure the effects of PCR annealing temperature and the ratio of host to bacterial DNA on the outcome of microbiota analysis, using pig microbiota as a model environment. FINDINGS: Six samples were chosen with an anticipated range of ratios of pig to bacterial genomic DNA, and universal cpn60 PCR amplification with an annealing temperature gradient was used to create libraries for pyrosequencing, resulting in 426,477 sequences from the six samples. The sequences obtained were classified as target (cpn60) or non-target based on the percent identity of their closest match to the cpnDB reference database, and target sequences were further processed to create microbiome profiles for each sample at each annealing temperature. Annealing temperature affected the amount of PCR product generated, with more product generated at higher temperatures. Samples containing proportionally more host genomic DNA yielded more non-target reads, especially at lower annealing temperatures. However, microbiome composition for each sample across the annealing temperature gradient remained consistent at both the phylum and operational taxonomic unit levels. Although some microbial sequences were detected at only one annealing temperature, these sequences accounted for a minority of the total microbiome. CONCLUSIONS: These results indicate that PCR annealing temperature does have an affect on cpn60 based microbiome profiles, but that most of the differences are due to differences in detection of low abundance sequences. Higher annealing temperatures resulted in larger amounts of PCR product and lower amounts of non-target sequence amplification, especially in samples containing proportionally large amounts of host DNA. Taken together these results provide important information to guide decisions about experimental design for cpn60 based microbiome studies.

Identification and Validation of Novel Hedgehog-Responsive Enhancers Predicted by Computational Analysis of Ci/Gli Binding Site Density.

The Hedgehog (Hh) signaling pathway directs a multitude of cellular responses during embryogenesis and adult tissue homeostasis. Stimulation of the pathway results in activation of Hh target genes by the transcription factor Ci/Gli, which binds to specific motifs in genomic enhancers. In Drosophila, only a few enhancers (patched, decapentaplegic, wingless, stripe, knot, hairy, orthodenticle) have been shown by in vivo functional assays to depend on direct Ci/Gli regulation. All but one (orthodenticle) contain more than one Ci/Gli site, prompting us to directly test whether homotypic clustering of Ci/Gli binding sites is sufficient to define a Hh-regulated enhancer. We therefore developed a computational algorithm to identify Ci/Gli clusters that are enriched over random expectation, within a given region of the genome. Candidate genomic regions containing Ci/Gli clusters were functionally tested in chicken neural tube electroporation assays and in transgenic flies. Of the 22 Ci/Gli clusters tested, seven novel enhancers (and the previously known patched enhancer) were identified as Hh-responsive and Ci/Gli-dependent in one or both of these assays, including: Cuticular protein 100A (Cpr100A); invected (inv), which encodes an engrailed-related transcription factor expressed at the anterior/posterior wing disc boundary; roadkill (rdx), the fly homolog of vertebrate Spop; the segment polarity gene gooseberry (gsb); and two previously untested regions of the Hh receptor-encoding patched (ptc) gene. We conclude that homotypic Ci/Gli clustering is not sufficient information to ensure Hh-responsiveness; however, it can provide a clue for enhancer recognition within putative Hedgehog target gene loci.

Adolescent suicide prevention in a school setting: use of a gatekeeper program.

Suicide is a major public health problem. Nationally, suicide is the third leading cause of death for adolescents. The purpose of this quality improvement project was to initiate and evaluate a gatekeeper suicide-prevention program within a local school system targeting faculty and staff without a medical or psychology background who interact regularly with middle- and high-school students. Following the implementation of this program, evaluation of increased knowledge related to adolescent suicide prevention was completed. All participants completed a pretest and posttest, and results indicate that the staff members' knowledge about identification of risk factors, behavioral responses to suicidal students, and knowledge of community resources were increased. This project highlights the need for planned and sustainable education and training for faculty and school staff who regularly interact with adolescents. Additionally, the importance of continued monitoring, training, and advocating for suicide prevention programming is noted.

Reverse-engineering a transcriptional enhancer: a case study in Drosophila.

Enhancers, or cis-regulatory elements, are the principal determinants of spatiotemporal patterning of gene expression. For reasons of clinical and research utility, it is desirable to build customized enhancers that drive novel gene expression patterns, but currently, we largely rely on "found" genomic elements. Synthetic enhancers, assembled from transcription factor binding sites taken from natural signal-regulated enhancers, generally fail to behave like their wild-type counterparts when placed in transgenic animals, suggesting that important aspects of enhancer function are still unexplored. As a step toward the creation of a truly synthetic regulatory element, we have undertaken an extensive structure-function study of an enhancer of the Drosophila decapentaplegic (dpp) gene that drives expression in the developing visceral mesoderm (VM). Although considerable past efforts have been made to dissect the dppVM enhancer, transgenic experiments presented here indicate that its activity cannot be explained by the known regulators alone. dppVM contains multiple, previously uncharacterized, regulatory sites, some of which exhibit functional redundancy. The results presented here suggest that even the best-studied enhancers must be further dissected before they can be fully understood, and before faithful synthetic elements based on them can be created. Implications for developmental genetics, mathematical modeling, and therapeutic applications are discussed.

Hsc70 focus formation at the periphery of HSV-1 transcription sites requires ICP27.

BACKGROUND: The cellular chaperone protein Hsc70, along with components of the 26S proteasome and ubiquitin-conjugated proteins have been shown to be sequestered in discrete foci in the nuclei of herpes simplex virus 1 (HSV-1) infected cells. We recently reported that cellular RNA polymerase II (RNAP II) undergoes proteasomal degradation during robust HSV-1 transcription, and that the immediate early protein ICP27 interacts with the C-terminal domain and is involved in the recruitment of RNAP II to viral transcription/replication compartments. METHODOLOGY/PRINCIPLE FINDINGS: Here we show that ICP27 also interacts with Hsc70, and is required for the formation of Hsc70 nuclear foci. During infection with ICP27 mutants that are unable to recruit RNAP II to viral replication sites, viral transcript levels were greatly reduced, viral replication compartments were poorly formed and Hsc70 focus formation was curtailed. Further, a dominant negative Hsc70 mutant that cannot hydrolyze ATP, interfered with RNAP II degradation during HSV-1 infection, and an increase in ubiquitinated forms of RNAP II was observed. There was also a decrease in virus yields, indicating that proteasomal degradation of stalled RNAP II complexes during robust HSV-1 transcription and replication benefits viral gene expression. CONCLUSIONS/SIGNIFICANCE: We propose that one function of the Hsc70 nuclear foci may be to serve to facilitate the process of clearing stalled RNAP II complexes from viral genomes during times of highly active transcription.

ICP27 interacts with the C-terminal domain of RNA polymerase II and facilitates its recruitment to herpes simplex virus 1 transcription sites, where it undergoes proteasomal degradation during infection.

Herpes simplex virus 1 (HSV-1) ICP27 has been shown to interact with RNA polymerase II (RNAP II) holoenzyme. Here, we show that ICP27 interacts with the C-terminal domain (CTD) of RNAP II and that ICP27 mutants that cannot interact fail to relocalize RNAP II to viral transcription sites, suggesting a role for ICP27 in RNAP II recruitment. Using monoclonal antibodies specific for different phosphorylated forms of the RNAP II CTD, we found that the serine-2 phosphorylated form, which is found predominantly in elongating complexes, was not recruited to viral transcription sites. Further, there was an overall reduction in phosphoserine-2 staining. Western blot analysis revealed that there was a pronounced decrease in the phosphoserine-2 form and in overall RNAP II levels in lysates from cells infected with wild-type HSV-1. There was no appreciable difference in cdk9 levels, suggesting that protein degradation rather than dephosphorylation was occurring. Treatment of infected cells with proteasome inhibitors MG-132 and lactacystin prevented the decrease in the phosphoserine-2 form and in overall RNAP II levels; however, there was a concomitant decrease in the levels of several HSV-1 late proteins and in virus yield. Proteasomal degradation has been shown to resolve stalled RNAP II complexes at sites of DNA damage to allow 3' processing of transcripts. Thus, we propose that at later times of infection when robust transcription and DNA replication are occurring, elongating complexes may collide and proteasomal degradation may be required for resolution.