Transient expression of a luciferase mRNA in plant-parasitic and free-living nematodes by electroporation.

Despite their economic significance in agricultural cropping systems, a lack of suitable molecular tools for manipulating gene expression has hindered progress in the functional genomics of plant parasitic nematodes (PPN). Obligate sexual reproduction and the obligate nature of PPN-host interactions further complicate the development of in vivo gene delivery and expression systems in these pests. Methods such as microinjection and microprojectile bombardment have been developed for introducing gene constructs into the free-living nematode, Caenorhabditis elegans. However, these procedures can be laborious and inefficient. Electroporation has been used extensively to introduce macromolecules, including single-stranded RNAs, into eukaryotic and prokaryotic cells. The technique has also been used for the delivery of DNA and double-stranded RNA constructs into nematodes by whole-animal electroporation. Here, we describe methods for the expression of a nematode-optimized NanoLuc luciferase mRNA in the form of in vitro transcripts following whole-animal electroporation of Heterodera glycines, Meloidogyne incognita, and C. elegans. The ability to transiently express single-stranded RNA constructs in economically important PPN provides a rapid means to evaluate nematode and/or foreign genes for their biological significance and potential role in nematode management.

Discovery of a Novel Member of the Carlavirus Genus from Soybean (Glycine max L. Merr.).

A novel member of the Carlavirus genus, provisionally named soybean carlavirus 1 (SCV1), was discovered by RNA-seq analysis of randomly collected soybean leaves in Illinois, USA. The SCV1 genome contains six open reading frames that encode a viral replicase, triple gene block proteins, a coat protein (CP) and a nucleic acid binding protein. The proteins showed highest amino acid sequence identities with the corresponding proteins of red clover carlavirus A (RCCVA). The predicted amino acid sequence of the SCV1 replicase was only 60.6% identical with the replicase of RCCVA, which is below the demarcation criteria for a new species in the family Betaflexiviridae. The predicted replicase and CP amino acid sequences of four SCV1 isolates grouped phylogenetically with those of members of the Carlavirus genus in the family Betaflexiviridae. The features of the encoded proteins, low nucleotide and amino acid sequence identities of the replicase with the closest member, and the phylogenetic grouping suggest SCV1 is a new member of the Carlavirus genus.

A pathogenesis-related protein GmPR08-Bet VI promotes a molecular interaction between the GmSHMT08 and GmSNAP18 in resistance to Heterodera glycines.

Soybean cyst nematode (SCN, Heterodera glycines) is the most devastating pest affecting soybean production worldwide. SCN resistance requires both the GmSHMT08 and the GmSNAP18 in 'Peking'-type resistance. Here, we describe the molecular interaction between GmSHMT08 and GmSNAP18, which is potentiated by a pathogenesis-related protein GmPR08-Bet VI. Like GmSNAP18 and GmSHMT08, GmPR08-Bet VI expression was induced in response to SCN and its overexpression decreased SCN cysts by 65% in infected transgenic soybean roots. Overexpression of GmPR08-Bet VI did not have an effect on SCN resistance when the two cytokinin-binding sites in GmPR08-Bet VI were mutated, indicating a new role of GmPR08-Bet VI in SCN resistance. GmPR08-Bet VI was mapped to a QTL for resistance to SCN using different mapping populations. GmSHMT08, GmSNAP18 and GmPR08-Bet VI localize to the cytosol and plasma membrane. GmSNAP18 expression and localization hyper-accumulated at the plasma membrane and was specific to the root cells surrounding the nematode in SCN-resistant soybeans. Genes encoding key components of the salicylic acid signalling pathway were induced under SCN infection. GmSNAP18 and GmPR08-Bet VI were also induced under salicylic acid and cytokinin exogenous treatments, while GmSHMT08 was induced only when the resistant GmSNAP18 was present, pointing to the presence of a molecular crosstalk between SCN-resistant genes and defence genes. Expression analysis of GmSHMT08 and GmSNAP18 identified the need of a minimum expression requirement to trigger the SCN resistance reaction. These results provide insight into a new response mechanism towards plant nematode resistance involving haplotype compatibility, gene dosage and hormone signalling.

Aphis glycines virus 1, a new bicistronic virus with two functional internal ribosome entry sites, is related to a group of unclassified viruses in the Picornavirales.

A novel picorna-like virus, provisionally named Aphis glycines virus 1 (ApGlV1) was discovered by high-throughput sequencing of soybean total RNAs and detected in suction trap-collected Aphis glycines. The ApGlV1 genome contains two large ORFs organized similar to those of dicipiviruses in the Picornaviridae where ORFs 1 and 2 encode structural and nonstructural proteins, respectively. Both ORFs are preceded by internal ribosome entry site (IRES) elements. The 5' IRES was more active in dual luciferase activity assays than the IRES in the intergenic region. The ApGlV1 genome was predicted to encode a serine protease instead of a cysteine protease and showed very low aa sequence identities to recognized members of the Picornavirales. In phylogenetic analyses based on capsid protein and RNA-dependent RNA polymerase sequences, ApGlV1 consistently clustered with a group of unclassified bicistronic picorna-like viruses discovered from arthropods and plants that may represent a novel family in the order Picornavirales.

Pathotypes Detected Among Populations of Pratylenchus neglectus Collected From Montana.

The root lesion nematode, Pratylenchus neglectus, is one of the most damaging nematodes to affect wheat worldwide. The nematode is widely distributed in Montana, primarily affecting winter wheat within the state. Managing the nematode primarily involves rotation to resistant and moderately resistant crops (peas, lentils, and barley). A nematode survey was conducted across the state nearly 10 years after an initial survey, to reassess the nematode threat and assess the impact of changing trends in crop rotations. To assess the broad applicability of rotation crops to control P. neglectus across Montana, greenhouse trials were conducted to challenge rotational crops using eight populations of P. neglectus collected from geographically diverse locations across the state. In the trials, conducted with four Montana crops, a significant interaction was detected between crop and nematode population (analysis of variance P < 0.001). Populations from Hill, Dawson, and Chouteau counties were found to be pathogenic on barley. Male nematodes were detected in seven of the eight pot culture populations, and these were confirmed to be P. neglectus by morphological and molecular methods. These results suggest a re-evaluation of barley and lentils as a management option for P. neglectus in Montana, as pathotypes for each exist within the state.

Transcriptome Profiling and Genome-Wide Association Studies Reveal GSTs and Other Defense Genes Involved in Multiple Signaling Pathways Induced by Herbicide Safener in Grain Sorghum.

Herbicide safeners protect cereal crops from herbicide injury by inducing genes and proteins involved in detoxification reactions, such as glutathione S-transferases (GSTs) and cytochrome P450s (P450s). Only a few studies have characterized gene or protein expression profiles for investigating plant responses to safener treatment in cereal crops, and most transcriptome analyses in response to safener treatments have been conducted in dicot model species that are not protected by safener from herbicide injury. In this study, three different approaches were utilized in grain sorghum (Sorghum bicolor (L.) Moench) to investigate mechanisms involved in safener-regulated signaling pathways. An initial transcriptome analysis was performed to examine global gene expression in etiolated shoot tissues of hybrid grain sorghum following treatment with the sorghum safener, fluxofenim. Most upregulated transcripts encoded detoxification enzymes, including P450s, GSTs, and UDP-dependent glucosyltransferases (UGTs). Interestingly, several of these upregulated transcripts are similar to genes involved with the biosynthesis and recycling/catabolism of dhurrin, an important chemical defense compound, in these seedling tissues. Secondly, 761 diverse sorghum inbred lines were evaluated in a genome-wide association study (GWAS) to determine key molecular-genetic factors governing safener-mediated signaling mechanisms and/or herbicide detoxification. GWAS revealed a significant single nucleotide polymorphism (SNP) associated with safener-induced response on chromosome 9, located within a phi-class SbGST gene and about 15-kb from a different phi-class SbGST. Lastly, the expression of these two candidate SbGSTs was quantified in etiolated shoot tissues of sorghum inbred BTx623 in response to fluxofenim treatment. SbGSTF1 and SbGSTF2 transcripts increased within 12-hr after fluxofenim treatment but the level of safener-induced expression differed between the two genes. In addition to identifying specific GSTs potentially involved in the safener-mediated detoxification pathway, this research elucidates a new direction for studying both constitutive and inducible mechanisms for chemical defense in cereal crop seedlings.

Active and inactive forms of biotin synthase occur in Heterodera glycines.

Heterodera glycines, the soybean cyst nematode (SCN), is a plant-parasitic nematode capable of manipulating host plant biochemistry and development. Many studies have suggested that the nematode has acquired genes from bacteria via horizontal gene transfer events (HGTs) that have the potential to enhance nematode parasitism. A recent allelic imbalance analysis identified two candidate virulence genes, which also appear to have entered the SCN genome through HGTs. One of the candidate genes, H. glycines biotin synthase (HgBioB), contained sequence polymorphisms between avirulent and virulent inbred SCN strains. To test the function of these HgBioB alleles, a complementation experiment using biotin synthase-deficient Escherichia coli was conducted. Here, we report that avirulent nematodes produce an active biotin synthase while virulent ones contain an inactive form of the enzyme. Moreover, sequencing analysis of HgBioB genes from SCN field populations indicates the presence of diverse mixture of HgBioB alleles with the virulent form being the most prevalent. We hypothesize that the mutations in the inactive HgBioB allele within the virulent SCN could result in a change in protein function that in some unknown way bolster its parasitic lifestyle.

An Agent-Based Metapopulation Model Simulating Virus-Based Biocontrol of Heterodera Glycines.

With recently discovered soybean cyst nematode (SCN) viruses, biological control of the nematodes is a theoretical possibility. This study explores the question of what kinds of viruses would make useful biocontrol agents, taking into account evolutionary and population dynamics. An agent-based model, Soybean Cyst Nematode Simulation (SCNSim), was developed to simulate within-host virulence evolution in a virus-nematode-soybean ecosystem. SCNSim was used to predict nematode suppression under a range of viral mutation rates, initial virulences, and release strategies. The simulation model suggested that virus-based biocontrol worked best when the nematodes were inundated with the viruses. Under lower infection prevalence, the viral burden thinned out rapidly due to the limited mobility and high reproductive rate of the SCN. In accordance with the generally accepted trade-off theory, SCNSim predicted the optimal initial virulence for the maximum nematode suppression. Higher initial virulence resulted in shorter lifetime transmission, whereas viruses with lower initial virulence values evolved toward avirulence. SCNSim also indicated that a greater viral mutation rate reinforced the virulence pathotype, suggesting the presence of a virulence threshold necessary to achieve biocontrol against SCN.

Immobility in the sedentary plant-parasitic nematode H. glycines is associated with remodeling of neuromuscular tissue.

The sedentary plant-parasitic nematodes are considered among the most economically damaging pathogens of plants. Following infection and the establishment of a feeding site, sedentary nematodes become immobile. Loss of mobility is reversed in adult males while females never regain mobility. The structural basis for this change in mobility is unknown. We used a combination of light and transmission electron microscopy to demonstrate cell-specific muscle atrophy and sex-specific renewal of neuromuscular tissue in the sedentary nematode Heterodera glycines. We found that both females and males undergo body wall muscle atrophy and loss of attachment to the underlying cuticle during immobile developmental stages. Male H. glycines undergo somatic muscle renewal prior to molting into a mobile adult. In addition, we found developmental changes to the organization and number of motor neurons in the ventral nerve cord correlated with changes in mobility. To further examine neuronal changes associated with immobility, we used a combination of immunohistochemistry and molecular biology to characterize the GABAergic nervous system of H. glycines during mobile and immobile stages. We cloned and confirmed the function of the putative H. glycines GABA synthesis-encoding gene hg-unc-25 using heterologous rescue in C. elegans. We found a reduction in gene expression of hg-unc-25 as well as a reduction in the number of GABA-immunoreactive neurons during immobile developmental stages. Finally, we found evidence of similar muscle atrophy in the phylogenetically diverged plant-parasitic nematode Meloidogyne incognita. Together, our data demonstrate remodeling of neuromuscular structure and function during sedentary plant-parasitic nematode development.

Genome Sequence of the Soybean Cyst Nematode (Heterodera glycines) Endosymbiont "Candidatus Cardinium hertigii" Strain cHgTN10.

In this study, we present the genome sequence of the "Candidatus Cardinium hertigii" strain cHgTN10, an endosymbiotic bacterium of the plant-parasitic nematode Heterodera glycines This is the first genome assembly reported for an endosymbiont directly sequenced from a tylenchid nematode.

Characterization of the Soluble NSF Attachment Protein gene family identifies two members involved in additive resistance to a plant pathogen.

Proteins with Tetratricopeptide-repeat (TPR) domains are encoded by large gene families and distributed in all plant lineages. In this study, the Soluble NSF-Attachment Protein (SNAP) subfamily of TPR containing proteins is characterized. In soybean, five members constitute the SNAP gene family: GmSNAP18, GmSNAP11, GmSNAP14, GmSNAP02, and GmSNAP09. Recently, GmSNAP18 has been reported to mediate resistance to soybean cyst nematode (SCN). Using a population of recombinant inbred lines from resistant and susceptible parents, the divergence of the SNAP gene family is analysed over time. Phylogenetic analysis of SNAP genes from 22 diverse plant species showed that SNAPs were distributed in six monophyletic clades corresponding to the major plant lineages. Conservation of the four TPR motifs in all species, including ancestral lineages, supports the hypothesis that SNAPs were duplicated and derived from a common ancestor and unique gene still present in chlorophytic algae. Syntenic analysis of regions harbouring GmSNAP genes in soybean reveals that this family expanded from segmental and tandem duplications following a tetraploidization event. qRT-PCR analysis of GmSNAPs indicates a co-regulation following SCN infection. Finally, genetic analysis demonstrates that GmSNAP11 contributes to an additive resistance to SCN. Thus, GmSNAP11 is identified as a novel minor gene conferring resistance to SCN.

Biochemical characterization of metabolism-based atrazine resistance in Amaranthus tuberculatus and identification of an expressed GST associated with resistance.

Rapid detoxification of atrazine in naturally tolerant crops such as maize (Zea mays) and grain sorghum (Sorghum bicolor) results from glutathione S-transferase (GST) activity. In previous research, two atrazine-resistant waterhemp (Amaranthus tuberculatus) populations from Illinois, U.S.A. (designated ACR and MCR), displayed rapid formation of atrazine-glutathione (GSH) conjugates, implicating elevated rates of metabolism as the resistance mechanism. Our main objective was to utilize protein purification combined with qualitative proteomics to investigate the hypothesis that enhanced atrazine detoxification, catalysed by distinct GSTs, confers resistance in ACR and MCR. Additionally, candidate AtuGST expression was analysed in an F2 population segregating for atrazine resistance. ACR and MCR showed higher specific activities towards atrazine in partially purified ammonium sulphate and GSH affinity-purified fractions compared to an atrazine-sensitive population (WCS). One-dimensional electrophoresis of these fractions displayed an approximate 26-kDa band, typical of GST subunits. Several phi- and tau-class GSTs were identified by LC-MS/MS from each population, based on peptide similarity with GSTs from Arabidopsis. Elevated constitutive expression of one phi-class GST, named AtuGSTF2, correlated strongly with atrazine resistance in ACR and MCR and segregating F2 population. These results indicate that AtuGSTF2 may be linked to a metabolic mechanism that confers atrazine resistance in ACR and MCR.

Molecular characterization of a new soybean-infecting member of the genus Nepovirus identified by high-throughput sequencing.

The complete nucleotide sequence of a new soybean-infecting member of the genus Nepovirus (provisionally named "soybean latent spherical virus" [SLSV]) was identified by high-throughput sequencing of RNAs from soybean leaf samples from North Dakota, USA. The sequences of RNAs 1 (8,190 nt) and 2 (5,788 nt) were completed by rapid amplification of cDNA ends. Each contained a single long open reading frame and a 3' nontranslated region of greater than 1,500 nt. The predicted amino acid sequences of the two ORFs were most closely related to nepoviruses in subgroup C. Full-length cDNAs of RNAs 1 and 2 were cloned and used to inoculate soybean plants, which did not display obvious symptoms. These results suggest that SLSV represents a new species in the genus Nepovirus.

A novel high-throughput multi-parameter flow cytometry based method for monitoring and rapid characterization of microbiome dynamics in anaerobic systems.

A novel multidimensional flow cytometry based method has been demonstrated to monitor and rapidly characterize the dynamics of the complex anaerobic microbiome associated with perturbations in external environmental factors. While community fingerprinting provides an estimate of the meta genomic structure, flow cytometry provides a fingerprint of the community morphology including its autofluorescence spectrum in a high-throughput manner. Using anaerobic microbial consortia perturbed with the controlled addition of various carbon sources, it is possible to quantitatively discriminate between divergent microbiome analogous to community fingerprinting techniques using automated ribosomal intergenic spacer analysis (ARISA). The utility of flow cytometry based method has also been demonstrated in a fully functional industry scale anaerobic digester to distinguish between microbiome composition caused by varying hydraulic retention time (HRT). This approach exploits the rich multidimensional information from flow cytometry for rapid characterization of the dynamics of microbial communities.

A SNARE-Like Protein and Biotin Are Implicated in Soybean Cyst Nematode Virulence.

Phytoparasitic nematodes that are able to infect and reproduce on plants that are considered resistant are referred to as virulent. The mechanism(s) that virulent nematodes employ to evade or suppress host plant defenses are not well understood. Here we report the use of a genetic strategy (allelic imbalance analysis) to associate single nucleotide polymorphisms (SNPs) with nematode virulence genes in Heterodera glycines, the soybean cyst nematode (SCN). To accomplish this analysis, a custom SCN SNP array was developed and used to genotype SCN F3-derived populations grown on resistant and susceptible soybean plants. Three SNPs reproducibly showed allele imbalances between nematodes grown on resistant and susceptible plants. Two candidate SCN virulence genes that were tightly linked to the SNPs were identified. One SCN gene encoded biotin synthase (HgBioB), and the other encoded a bacterial-like protein containing a putative SNARE domain (HgSLP-1). The two genes mapped to two different linkage groups. HgBioB contained sequence polymorphisms between avirulent and virulent nematodes. However, the gene encoding HgSLP-1 had reduced copy number in virulent nematode populations and appears to produce multiple forms of the protein via intron retention and alternative splicing. We show that HgSLP-1 is an esophageal-gland protein that is secreted by the nematode during plant parasitism. Furthermore, in bacterial co-expression experiments, HgSLP-1 co-purified with the SCN resistance protein Rhg1 α-SNAP, suggesting that these two proteins physically interact. Collectively our data suggest that multiple SCN genes are involved in SCN virulence, and that HgSLP-1 may function as an avirulence protein and when absent it helps SCN evade host defenses.

Activation of the aryl hydrocarbon receptor during development enhances the pulmonary CD4+ T-cell response to viral infection.

Respiratory infections are a threat to health and economies worldwide, yet the basis for striking variation in the severity of infection is not completely understood. Environmental exposures during development are associated with increased severity and incidence of respiratory infection later in life. Many of these exposures include ligands of the aryl hydrocarbon receptor (AHR), a transcription factor expressed by immune and nonimmune cells. In adult animals, AHR activation alters CD4(+) T cells and changes immunopathology. Developmental AHR activation impacts CD4(+) T-cell responses in lymphoid tissues, but whether skewed responses are also present in the infected lung is unknown. To determine whether pulmonary CD4(+) T-cell responses are modified by developmental AHR activation, mice were exposed to the prototypical AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin during development and infected with influenza virus as adults. Lungs of exposed offspring had greater bronchopulmonary inflammation compared with controls, and activated, virus-specific CD4(+) T cells contributed to the infiltrating leukocytes. These effects were CD4(+) T cell subset specific, with increases in T helper type 1 and regulatory T cells, but no change in the frequency of T helper type 17 cells in the infected lung. This is in direct contrast to prior reports of suppressed conventional CD4(+) T-cell responses in the lymph node. Using adoptive transfers and manipulating the pathogen properties, we determined that developmental exposure influenced factors intrinsic and extrinsic to CD4(+) T cells and may involve developmentally induced changes in signals from infected lung epithelial cells. Thus developmental exposures lead to context-dependent changes in pulmonary CD4(+) T-cell subsets, which may contribute to differential responses to respiratory infection.

Comparative mapping of the wild perennial Glycine latifolia and soybean (G. max) reveals extensive chromosome rearrangements in the genus Glycine.

Soybean (Glycine max L. Mer.), like many cultivated crops, has a relatively narrow genetic base and lacks diversity for some economically important traits. Glycine latifolia (Benth.) Newell & Hymowitz, one of the 26 perennial wild Glycine species related to soybean in the subgenus Glycine Willd., shows high levels of resistance to multiple soybean pathogens and pests including Alfalfa mosaic virus, Heterodera glycines Ichinohe and Sclerotinia sclerotiorum (Lib.) de Bary. However, limited information is available on the genomes of these perennial Glycine species. To generate molecular resources for gene mapping and identification, high-density linkage maps were constructed for G. latifolia using single nucleotide polymorphism (SNP) markers generated by genotyping by sequencing and evaluated in an F2 population and confirmed in an F5 population. In each population, greater than 2,300 SNP markers were selected for analysis and segregated to form 20 large linkage groups. Marker orders were similar in the F2 and F5 populations. The relationships between G. latifolia linkage groups and G. max and common bean (Phaseolus vulgaris L.) chromosomes were examined by aligning SNP-containing sequences from G. latifolia to the genome sequences of G. max and P. vulgaris. Twelve of the 20 G. latifolia linkage groups were nearly collinear with G. max chromosomes. The remaining eight G. latifolia linkage groups appeared to be products of multiple interchromosomal translocations relative to G. max. Large syntenic blocks also were observed between G. latifolia and P. vulgaris. These experiments are the first to compare genome organizations among annual and perennial Glycine species and common bean. The development of molecular resources for species closely related to G. max provides information into the evolution of genomes within the genus Glycine and tools to identify genes within perennial wild relatives of cultivated soybean that could be beneficial to soybean production.

A novel flavivirus in the soybean cyst nematode.

Heterodera glycines, the soybean cyst nematode (SCN), is a subterranean root pathogen that causes the most damaging disease of soybean in the USA. A novel nematode virus genome, soybean cyst nematode virus 5 (SbCNV-5), was identified in RNA sequencing data from SCN eggs and second-stage juveniles. The SbCNV-5 RNA-dependent RNA polymerase and RNA helicase domains had homology to pestiviruses in the family Flaviviridae, suggesting that SbCNV-5 is a positive-polarity ssRNA virus. SbCNV-5 RNA was present in all nematode developmental stages, indicating a transovarial mode of transmission, but is also potentially sexually transmitted via the male. SbCNV-5 was common in SCN laboratory cultures and in nematode populations isolated from the field. Transmission electron microscopy of sections from a female SCN showed virus particles budding from the endoplasmic reticulum and in endosomes. The size of the viral genome was 19 191 nt, which makes it much larger than other known pestiviruses. Additionally, the presence of a methyltransferase in the SbCNV-5 genome is atypical for a pestivirus. When cDNA sequences were mapped to the genome of SbCNV-5, a disproportionate number aligned to the 3' NTR, suggesting that SbCNV-5 produces a subgenomic RNA, which was confirmed by RNA blot analysis. As subgenomic RNAs and methyltransferases do not occur in pestiviruses, we conclude that SbCNV-5 is a new flavivirus infecting SCNs.

Inflammation-induced interstitial migration of effector CD4⁺ T cells is dependent on integrin αV.

Leukocytes must traverse inflamed tissues to effectively control local infection. Although motility in dense tissues seems to be integrin independent and based on actomyosin-mediated protrusion and contraction, during inflammation, changes to the extracellular matrix (ECM) may necessitate distinct motility requirements. Indeed, we found that the interstitial motility of T cells was critically dependent on Arg-Gly-Asp (RGD)-binding integrins in the inflamed dermis. Inflammation-induced deposition of fibronectin was functionally linked to higher expression of integrin αV on effector CD4⁺ T cells. By intravital multiphoton imaging, we found that the motility of CD4⁺ T cells was dependent on αV expression. Selective blockade or knockdown of αV arrested T helper type 1 (TH1) cells in the inflamed tissue and attenuated local effector function. Our data demonstrate context-dependent specificity of lymphocyte movement in inflamed tissues that is essential for protective immunity.

Championing person-first language: a call to psychiatric mental health nurses.

At the heart of recovery-oriented psychiatric mental health care are the dignity and respect of each person and the ways in which helping professionals convey a person's uniqueness, strengths, abilities, and needs. "Person-first language" is a form of linguistic expression relying on words that reflect awareness, a sense of dignity, and positive attitudes about people with disabilities. As such, person-first language places emphasis on the person first rather than the disability (e.g., "person with schizophrenia" rather than "a schizophrenic"). This article champions the use of person-first language as a foundation for recovery-oriented practice and enhanced collaborative treatment environments that foster respect, human dignity, and hope.