Molecular convergence and positive selection associated with the evolution of symbiont transmission mode in stony corals.

Heritable symbioses have been critical for the evolution of life. The genetic consequences of evolving a heritable symbiosis from the perspective of the symbiont are well established, but concomitant changes in the host remain unresolved. In stony corals, heritable, vertical transmission has evolved repeatedly, providing a unique opportunity to investigate the genomic basis of this complex trait. We conducted a comparative analysis of 25 coral transcriptomes to identify orthologous genes exhibiting signatures of positive selection and convergent amino acid substitutions in vertically transmitting lineages. The frequency of convergence events tends to be higher among vertically transmitting lineages, consistent with the proposed role of selection in driving the evolution of convergent transmission mode phenotypes. Of 10 774 orthologous genes, 403 exhibited at least one molecular convergence event and evidence of positive selection in at least one vertically transmitting lineage. Functional enrichments among these top candidate genes include processes previously implicated in symbiosis including endocytosis, immune response, cytoskeletal protein binding and cytoplasmic membrane-bounded vesicles. Finally, several novel candidates were identified among 100 genes showing evidence of positive selection at the particular convergence event, highlighting the value of our approach for generating new insight into host mechanisms associated with the evolution of heritable symbioses.

Comparative transcriptomics of sympatric species of coral reef fishes (genus: Haemulon).

BACKGROUND: Coral reefs are major hotspots of diversity for marine fishes, yet there is still ongoing debate on the mechanisms that promote divergence in these rich ecosystems. Our understanding of how diversity originates in this environment could be enhanced by investigating the evolutionary dynamics of closely related fishes with overlapping ranges. Here, we focus on grunts of the genus Haemulon, a group of coral reef fishes with 15 species in the Western Atlantic, 11 of which are syntopic. METHODS: Wild fish samples from three sympatric species of the Caribbean: Haemulon flavolineatum, H. carbonarium and H. macrostomum, were collected while SCUBA diving. RNA was extracted from livers, and the transcriptomes were assembled and annotated to investigate positive selection (Pairwise d N/d S) and patterns of gene expression between the three species. RESULTS: Pairwise d N/d S analyses showed evidence of positive selection for genes associated with immune response, cranial morphology and formation of the anterior-posterior axis. Analyses of gene expression revealed that despite their sympatric distribution, H. macrostomum showed upregulation of oxidation-reduction machinery, while there was evidence for activation of immune response in H. carbonarium. DISCUSSION: Overall, our analyses suggest closely related grunts show important differences in genes associated with body shape and feeding morphology, a result in-line with previous morphological studies in the group. Further, despite their overlapping distribution they interact with their environment in distinct fashions. This is the largest compendium of genomic information for grunts thus far, representing a valuable resource for future studies in this unique group of coral reef fishes.

Antimicrobial and stress responses to increased temperature and bacterial pathogen challenge in the holobiont of a reef-building coral.

Global increases in coral disease prevalence have been linked to ocean warming through changes in coral-associated bacterial communities, pathogen virulence and immune system function. However, the interactive effects of temperature and pathogens on the coral holobiont are poorly understood. Here, we assessed three compartments of the holobiont (host, Symbiodinium and bacterial community) of the coral Montipora aequituberculata challenged with the pathogen Vibrio coralliilyticus and the commensal bacterium Oceanospirillales sp. under ambient (27°C) and elevated (29.5 and 32°C) seawater temperatures. Few visual signs of bleaching and disease development were apparent in any of the treatments, but responses were detected in the holobiont compartments. V. coralliilyticus acted synergistically and negatively impacted the photochemical efficiency of Symbiodinium at 32°C, while Oceanospirillales had no significant effect on photosynthetic efficiency. The coral, however, exhibited a minor response to the bacterial challenges, with the response towards V. coralliilyticus being significantly more pronounced, and involving the prophenoloxidase-activating system and multiple immune system-related genes. Elevated seawater temperatures did not induce shifts in the coral-associated bacterial community, but caused significant gene expression modulation in both Symbiodinium and the coral host. While Symbiodinium exhibited an antiviral response and upregulated stress response genes, M. aequituberculata showed regulation of genes involved in stress and innate immune response processes, including immune and cytokine receptor signalling, the complement system, immune cell activation and phagocytosis, as well as molecular chaperones. These observations show that M. aequituberculata is capable of maintaining a stable bacterial community under elevated seawater temperatures and thereby contributes to preventing disease development.

Evolutionary Consequences of DNA Methylation in a Basal Metazoan.

Gene body methylation (gbM) is an ancestral and widespread feature in Eukarya, yet its adaptive value and evolutionary implications remain unresolved. The occurrence of gbM within protein-coding sequences is particularly puzzling, because methylation causes cytosine hypermutability and hence is likely to produce deleterious amino acid substitutions. We investigate this enigma using an evolutionarily basal group of Metazoa, the stony corals (order Scleractinia, class Anthozoa, phylum Cnidaria). We show that patterns of coral gbM are similar to other invertebrate species, predicting wide and active transcription and slower sequence evolution. We also find a strong correlation between gbM and codon bias, resulting from systematic replacement of CpG bearing codons. We conclude that gbM has strong effects on codon evolution and speculate that this may influence establishment of optimal codons.

CORAL REEFS. Genomic determinants of coral heat tolerance across latitudes.

As global warming continues, reef-building corals could avoid local population declines through "genetic rescue" involving exchange of heat-tolerant genotypes across latitudes, but only if latitudinal variation in thermal tolerance is heritable. Here, we show an up-to-10-fold increase in odds of survival of coral larvae under heat stress when their parents come from a warmer lower-latitude location. Elevated thermal tolerance was associated with heritable differences in expression of oxidative, extracellular, transport, and mitochondrial functions that indicated a lack of prior stress. Moreover, two genomic regions strongly responded to selection for thermal tolerance in interlatitudinal crosses. These results demonstrate that variation in coral thermal tolerance across latitudes has a strong genetic basis and could serve as raw material for natural selection.

Bimodal signatures of germline methylation are linked with gene expression plasticity in the coral Acropora millepora.

BACKGROUND: In invertebrates, genes belonging to dynamically regulated functional categories appear to be less methylated than "housekeeping" genes, suggesting that DNA methylation may modulate gene expression plasticity. To date, however, experimental evidence to support this hypothesis across different natural habitats has been lacking. RESULTS: Gene expression profiles were generated from 30 pairs of genetically identical fragments of coral Acropora millepora reciprocally transplanted between distinct natural habitats for 3 months. Gene expression was analyzed in the context of normalized CpG content, a well-established signature of historical germline DNA methylation. Genes with weak methylation signatures were more likely to demonstrate differential expression based on both transplant environment and population of origin than genes with strong methylation signatures. Moreover, the magnitude of expression differences due to environment and population were greater for genes with weak methylation signatures. CONCLUSIONS: Our results support a connection between differential germline methylation and gene expression flexibility across environments and populations. Studies of phylogenetically basal invertebrates such as corals will further elucidate the fundamental functional aspects of gene body methylation in Metazoa.

Simultaneous detection of human mitochondrial DNA and nuclear-inserted mitochondrial-origin sequences (NumtS) using forensic mtDNA amplification strategies and pyrosequencing technology.

Next-generation sequencing technologies enable the identification of minor mitochondrial DNA variants with higher sensitivity than Sanger methods, allowing for enhanced identification of minor variants. In this study, mixtures of human mtDNA control region amplicons were subjected to pyrosequencing to determine the detection threshold of the Roche GS Junior(®) instrument (Roche Applied Science, Indianapolis, IN). In addition to expected variants, a set of reproducible variants was consistently found in reads from one particular amplicon. A BLASTn search of the variant sequence revealed identity to a segment of a 611-bp nuclear insertion of the mitochondrial control region (NumtS) spanning the primer-binding sites of this amplicon (Nature 1995;378:489). Primers (Hum Genet 2012;131:757; Hum Biol 1996;68:847) flanking the insertion were used to confirm the presence or absence of the NumtS in buccal DNA extracts from twenty donors. These results further our understanding of human mtDNA variation and are expected to have a positive impact on the interpretation of mtDNA profiles using deep-sequencing methods in casework.

A specific role of AGS3 in the surface expression of plasma membrane proteins.

Activator of G protein signaling 3 (AGS3), originally identified in a functional screen for mammalian proteins that activate heterotrimeric G protein signaling, is known to be involved in drug-seeking behavior and is up-regulated during cocaine withdrawal in animal models. These observations indicate a potential role for AGS3 in the formation or maintenance of neural plasticity. We have found that the overexpression of AGS3 alters the surface-to-total ratios of a subset of heterologously expressed plasma membrane receptors and channels. Further analysis of the endocytic trafficking of one such protein by a biotin-based internalization assay suggests that overexpression of AGS3 moderately affects the internalization or recycling of surface proteins. Moreover, AGS3 overexpression and siRNA-mediated knockdown of AGS3 both result in the dispersal of two endogenously expressed trans-Golgi network (TGN)-associated cargo proteins without influencing those in the cis- or medial-Golgi compartments. Finally, adding a TGN-localization signal to a CD4-derived reporter renders the trafficking of fusion protein sensitive to AGS3. Taken together, our data support a model wherein AGS3 modulates the protein trafficking along the TGN/plasma membrane/endosome loop.

Identification of risk factors for increased cost, charges, and length of stay for cardiac patients.

BACKGROUND: In this study we explored different risk model options to provide clinicians with predictions for resource utilization. The hypotheses were that predictors of mortality are not predictive of resource consumption, and that there is a correlation between cost estimates derived using a cost-to-charge ratio or a product-line costing approach. METHODS: From March 1992 to June 1995, 2,481 University of Colorado Hospital patients admitted for ischemic heart disease were classified by diagnosis-related group code as having undergone or experienced coronary bypass procedures (CBP), percutaneous cardiovascular procedures (PCVP), acute myocardial infarction (AMI), and other cardiac-related discharges (Other). For each diagnosis-related group, Cox proportional hazards models were developed to determine predictors of cost, charges, and length of stay. RESULTS: The diagnosis groups differed in the clinical factors that predicted resource use. As the two costing methods were highly correlated, either approach may be used to assess relative resource consumption provided costs are reconciled to audited financial statements. CONCLUSIONS: To develop valid prediction models for costs of care, the clinical risk factors that are traditionally used to predict risk-adjusted mortality may need to be expanded.

War trauma experience and behavioral screening of Bosnian refugee children resettled in Massachusetts.

The authors assessed war violence exposure and behavioral symptoms in Bosnian refugee children in Massachusetts and the utility of behavioral screening of refugees during the Refugee Health Assessment (RHA), required of newly arrived refugees. The study was a survey of 31 Bosnian refugee children in 1996 at the International Clinic of Boston Medical Center, the state's largest contracted provider of the RHA. Subjects were also offered referrals to appropriate mental health services. Sixty-eight percent experienced long-term separation from a parent. Eighty-one percent were directly exposed to armed combat. Seventy-one percent experienced the death of a close friend or relative. Fifty-two percent experienced economic deprivation. Families reported behavioral symptoms for 77% of children. Only one family expressed interest in psychosocial services of any kind. Large numbers of Bosnian refugees are likely to have experienced traumatic war violence and are at risk of behavioral symptoms. The RHA affords opportunities to screen for behavioral problems but not to intervene. Primary care providers and other clinicians should be aware of likely recurrences of symptoms in high-risk children such as these.

Cutaneous and systemic responses during primary and challenge infestations of sheep with the sheep scab mite, Psoroptes ovis.

Lesional area was estimated and lesional mast cells and eosinophils were counted during primary and challenge infestations of sheep with Psoroptes ovis. In addition, circulating basophils and eosinophils and serum P. ovis-specific immunoglobulin IgE antibody were quantified. Expansion of lesional area was significantly less and serum IgE titres significantly greater in challenge than primary infestations. Lesional mast cell hyperplasia and massive eosinophil infiltration accompanied by raised titres of P. ovis antigen-specific IgE antibody were compatible with an IgE-mediated Type-1 allergic reaction, while detection of lesional basophils was suggestive of cutaneous basophil hypersensitivity. The temporal pattern of lesional mastocytosis and eosinophil infiltration and the role of these cells and serum IgE in the immune response is discussed.

Response rates to surveys with self-addressed, stamped envelopes versus a self-addressed label.

Return rates were studied for a survey on the perceived professional preparation of therapeutic recreation specialists by directors of college and university recreational therapy programs. Specifically, returns for surveys with self-addressed, stamped return envelopes enclosed were compared to those with self-adhering return address labels. Of the 75 surveys sent with a self-addressed, stamped return envelope, 42 responses (56%) were returned at a cost of $2.58 per respondent. Of the 72 surveys sent with a self-adhering return address label 43 responses (59.7%) were returned at a cost of $1.56 per response. While there was no significant difference in return rates, the self-adhering return address label was more cost effective than the self-addressed, stamped, return envelopes.

Computerized left ventricular pressure-volume relationships (pV-loops) using disposable angiographic tip transducer pigtail catheters.

Left ventricular pressure-volume relationships expressed as pV loops could yield important hemodynamic information in the cardiac catheterization laboratory. Many clinical situations might benefit from a quantitative assessment of left ventricular function. Potential applications of pV loops include the assessment of vasoactive and inotropic drugs, balloon valvuloplasty, coronary angioplasty, and surgical treatment of valvular heart disease. For many years the clinical use of pV loops has been hindered by logistical difficulties. The ability to merge on-line concurrent digital imaging data for computation of left ventricular volume and digital left ventricular pressure wave forms obtained from high fidelity tip-transducer angiocatheters has allowed us to develop a technique which can generate pV loops during cardiac catheterization procedures. The method offers an automated measurement of left ventricular volume independent of edge detection or an interactive technique for tracing endocardial borders by a trained operator. Illustrative case studies are included to demonstrate the potential of the method during ventricular angiographic procedures. Implementation and computational time requirements of the method are discussed. The concept and the value of pV loop generation to study left ventricular performance has been known for many years. Combining digital imaging and digital physiologic data obtained with disposable tip-transducer angiocatheters with modern networking technology, the technique can more easily be applied to catheterization procedures and could enhance invasive hemodynamic assessment of left ventricular function.

Continuous intravenous epoprostenol for pulmonary hypertension due to the scleroderma spectrum of disease. A randomized, controlled trial.

BACKGROUND: Pulmonary hypertension is a progressive and often fatal complication of the scleroderma spectrum of disease for which no treatment has been proven effective in a randomized trial. OBJECTIVE: To determine the effect of epoprostenol on pulmonary hypertension secondary to the scleroderma spectrum of disease. DESIGN: Randomized, open-label, controlled trial. SETTING: 17 pulmonary hypertension referral centers. PATIENTS: 111 patients with moderate to severe pulmonary hypertension. INTERVENTION: Epoprostenol plus conventional therapy or conventional therapy alone. MEASUREMENTS: The primary outcome measure was exercise capacity. Other measures were cardiopulmonary hemodynamics, signs and symptoms of pulmonary hypertension and scleroderma, and survival. RESULTS: Exercise capacity improved with epoprostenol (median distance walked in 6 minutes, 316 m at 12 weeks compared with 270 m at baseline) but decreased with conventional therapy (192 m at 12 weeks compared with 240 m at baseline). The difference between treatment groups in the median distance walked at week 12 was 108 m (95% CI, 55.2 m to 180.0 m) (P < 0.001). Hemodynamics improved at 12 weeks with epoprostenol. The changes in mean pulmonary artery pressure for the epoprostenol and conventional therapy groups were -5.0 and 0.9 mm Hg, respectively (difference, -6.0 mm Hg [CI, -9.0 to -3.0 mm Hg), and the mean changes in pulmonary vascular resistance were -4.6 and 0.9 mm Hg/L per minute, respectively (difference, -5.5 mm Hg/L per minute [CI, -7.3 to -3.7 mm Hg/L per minute). Twenty-one patients treated with epoprostenol and no patients receiving conventional therapy showed improved New York Heart Association functional class. Borg Dyspnea Scores and Dyspnea-Fatigue Ratings improved in the epoprostenol group. Trends toward greater improvement in severity of the Raynaud phenomenon and fewer new digital ulcers were seen in the epoprostenol group. Four patients in the epoprostenol group and five in the conventional therapy group died (P value not significant). Side effects of epoprostenol therapy included jaw pain, nausea, and anorexia. Adverse events related to the epoprostenol delivery system included sepsis, cellulitis, hemorrhage, and pneumothorax (4% incidence for each condition). CONCLUSIONS: Continuous epoprostenol therapy improves exercise capacity and cardiopulmonary hemodynamics in patients with pulmonary hypertension due to the scleroderma spectrum of disease.

Hyperuricemia in severe pulmonary hypertension.

STUDY OBJECTIVE: Hyperuricemia occurs frequently in patients with myeloproliferative and lymphoproliferative disorders and in patients with congenital heart disease associated with polycythemia. Whether hyperuricemia is common in patients with severe pulmonary hypertension is not known. DESIGN, PATIENTS, MEASUREMENTS: In the Pulmonary Hypertension Center at the University of Colorado Health Sciences Center between September 1991 and August 1997, 442 consecutive patients were evaluated with right heart catheterization; 191 patients also had a measurement of the serum uric acid (UA) in close temporal proximity to the hemodynamic evaluation. RESULTS: Of the 191 patients with a complete data set, 99 patients had primary pulmonary hypertension (PPH) and 92 had secondary pulmonary hypertension. For the entire cohort with severe pulmonary hypertension (n = 191), there was a positive correlation between the natural logarithm of the serum UA (lnUA) and the mean right atrial pressure (RAP; r = 0.47; p < 0.001). When analyzed separately, the correlation between lnUA and RAP was stronger in the patients with PPH (r = 0.642; p < 0.001). This correlation cannot be explained by diuretic use or impaired hepatocellular function. Neither mean pulmonary artery pressure nor cardiac output was as well correlated with the RAP when compared with the lnUA. Some patients with PPH had serum UA measurements repeated during treatment with chronic IV prostacyclin infusion. Eleven of these 18 patients (61%) demonstrated a decrease in serum UA during prostacyclin treatment. CONCLUSION: There is a positive correlation between the RAP elevation and the serum UA levels in patients with PPH. Serum UA levels drop in some, but not all PPH patients during chronic prostacyclin infusion therapy.

3D coronary reconstruction from routine single-plane coronary angiograms: clinical validation and quantitative analysis of the right coronary artery in 100 patients.

BACKGROUND: Current coronary angiographic techniques display complex three-dimensional (3D) coronary structures in two dimensions (2D). We have developed a 3D reconstruction (3DR) algorithm using standard single-plane angiographic images that allows for 3D display of coronary structures. The purpose of this study was to validate our 3DR algorithm and quantify anatomic characteristics of the right coronary artery (RCA) in vivo. METHODS: Accuracy and reproducibility studies were performed using 3DRs of a coronary phantom and in vivo following 3DRs in 40 patients. The anatomic features of the RCA were then quantified in 100 patients. RESULTS: Comparison of length and bifurcation angles (BA) from the phantom to the 3DRs revealed good accuracy and correlation for both (r = 0.95 and 0.93 respectively), with diameter error of < 7%. In vivo, the average root mean square (RMS) error in the spatial coordinates of the vessel centerlines was 3.12 +/- 0.77 and 3.16 +/- 0.75 mm in 20 left coronary arteries (LCA) and 20 RCAs respectively. Interobserver average RMS error was 3.47 +/- 1.96 mm and intraobserver average RMS error was 3.02 +/- 1.07 and 3.44 +/- 1.57 mm for two different operators (p = NS). The average RCA length was 10.2 +/- 1.7 cm, average radius of curvature (ROC) was 52 +/- 9 degrees, and the average 3D bifurcation angle of the posterior descending artery (PDA) from the RCA was 55 +/- 22 degrees. Foreshortening (FS) of the segments of the RCA in three 'standard' projections ranged from 0-60, 0-75, and 0-82% respectively. CONCLUSIONS: Using our 3DR algorithm patient-specific anatomic characteristics can be accurately displayed and quantified, expanding the information that can be derived from routine coronary angiography.

Restraining orders: a frequent marker of adverse maternal health.

OBJECTIVE: Women with histories of interpersonal violence are poorly identified because of barriers in self-disclosure. This study identified differences on maternal health and child behavior between women who report filing a restraining order (RO) and those who do not among a nonreferred sample of women living in high-crime neighborhoods. METHODS: During a maternal interview mothers were asked whether they ever filed a RO, the victim/defendant relationship, the number of times, and the year of the filing. Four types of violence were coded independently based on maternal narratives: verbal harassment, verbal threats or intimidation, physical assault, and destruction of property. We controlled for differences between RO and non-RO groups regarding demographic background, partner characteristics, other types of maternal past victimization, and use of alcohol and illicit drugs. Outcomes for mothers include partner aggression (Conflict Tactics Scale-R), perception of health and bodily pain (SF-36 Health Survey), distress symptoms (SCL90-R), posttraumatic stress (PTS)-related symptoms, and partial posttraumatic stress disorder (PTSD) diagnosis (modified Diagnostic Interview Schedule PTSD-Module). Outcomes for the child include partner aggression (Conflict Tactic Scale-R), behavior problems (CBC 2-3 or Child Behavior Checklist), and PTS-related symptoms (PTS-related symptom checklist). PATIENTS: One hundred sixty patients between 3.0 to 6.1 years who resided within five residential ZIP codes with a high rate of local crime in the City of Boston were drawn from a pediatric care clinic practice. Patients were participants of a larger study about the impact of community violence on mother-child relations. RESULTS: Sixty-four (40%) of 160 mothers reported a history of filing a RO against a current boyfriend or husband (39%), ex-boyfriend or husband (44%), someone known (8%), or other (9%), with a mean of 3.9 years (standard deviation = 3.5 years) since RO filing. After controlling for covariates of marital status, immigrant status, public assistance, and lifetime sexual victimization, we found a significant multivariate analysis of covariance group effect on maternal outcomes. Analysis of covariance analyses indicated that mothers in the RO group experienced higher current partner verbal aggression and physical violence to mother, poorer health, and higher PTS-related symptoms, compared with mothers in the non-RO group. More mothers in the RO group met partial lifetime PTSD diagnosis. Unadjusted for maternal covariates, the multivariate analysis of variance analyses on child outcomes (partner aggression to child, behavior problems, and PTS-related symptoms) indicated a nonsignificant group effect. CONCLUSIONS: Among dyads residing in high-risk crime areas, the incidence of RO histories is substantive considering this was a nonshelter, nonreferred sample. The inquiry about the history of a RO may provide a new and efficient marker to quality of current partner relationship, maternal health, and maternal stress-related symptomatology.

Continuous infusion of epoprostenol improves the net balance between pulmonary endothelin-1 clearance and release in primary pulmonary hypertension.

BACKGROUND: Primary pulmonary hypertension results from progressive narrowing of the precapillary pulmonary vasculature. A variety of endothelial abnormalities have been identified, including a net reduction in pulmonary clearance of the vasoconstrictor and smooth muscle mitogen endothelin-1. In many patients, net pulmonary release of endothelin-1 is observed. Chronic infusions of epoprostenol (prostacyclin) improve functional capacity, survival, and hemodynamics in patients with advanced primary pulmonary hypertension. We hypothesized that the epoprostenol infusions, as compared with conventional therapy, might alter the abnormal pulmonary endothelin-1 homeostasis. METHODS AND RESULTS: Using a subset of patients from a larger randomized study comparing epoprostenol plus conventional therapy (n=11 in the present study) with conventional therapy alone (n=7 in the present study), we determined the ratio of plasma endothelin-1 levels in systemic arterial blood leaving the lung to levels in mixed venous blood entering the lung both before randomization and after 88 days of continuous therapy. There were no differences between the 2 groups before therapy, but by day 88, the epoprostenol-treated group had a greater proportion of patients (82%) with an arterial/venous ratio <1 than did the conventional therapy group, in which only 29% of patients had a ratio <1 (P<0.05). CONCLUSIONS: These results suggest that continuous epoprostenol therapy may have a beneficial effect on the balance between endothelin-1 clearance and release in many patients with primary pulmonary hypertension and may provide one explanation for the salutary effect of epoprostenol in this disease.

Mental health services for children who witness domestic violence.

Exposure to domestic violence has significant negative repercussions for children's social, emotional, and academic functioning. In the past decade, mental health professionals have developed treatment programs and approaches aimed at mitigating these deleterious effects. Their efforts, however, are often hampered by difficulty identifying and gaining access to the target population because the occurrence of domestic violence remains a family secret in many households. Clinicians and researchers have published descriptions of group and individual therapy approaches for children who witness domestic violence. These approaches share several goals: promoting open discussion about children's experiences with domestic violence, helping children to deal with the emotions and consequences that follow such exposure, reducing the problematic symptoms children experience, strengthening children's relationships with their nonabusive caregivers, and helping children and their families to create and maintain relationships and living situations that are free from violence and abuse. One limitation of the literature describing these interventions is the absence of controlled outcome studies demonstrating the effects of these programs, in the short and long terms. Thus, development of such evaluative components is an important future direction for this field. Some of the other challenges that confront clinicians include working with children's families, addressing children's complex and intense emotional experiences, and determining whether children have themselves been victims of abuse or neglect (and then interfacing with child protective services).