Microbiome manipulation by corals and other Cnidaria via quorum quenching.

A dynamic mucous layer containing numerous micro-organisms covers the surface of corals and has multiple functions including both removal of sediment and "food gathering."1 It is likely to also act as the primary barrier to infection; various proteins and compounds with antimicrobial activity have been identified in coral mucus, though these are thought to be largely or exclusively of microbial origin. As in Hydra,2 anti-microbial peptides (AMPs) are likely to play major roles in regulating the microbiomes of corals.3,4 Some eukaryotes employ a complementary but less obvious approach to manipulate their associated microbiome by interfering with quorum signaling, effectively preventing bacteria from coordinating gene expression across a population. Our investigation of immunity in the reef-building coral Acropora millepora,5 however, led to the discovery of a coral gene referred to here as AmNtNH1 that can inactivate a range of acyl homoserine lactones (AHLs), common bacterial quorum signaling molecules, and is induced on immune challenge of adult corals and expressed during the larval settlement process. Closely related proteins are widely distributed within the Scleractinia (hard corals) and some other cnidarians, with multiple paralogs in Acropora, but their closest relatives are bacterial, implying that these are products of one or more lateral gene transfer events post-dating the cnidarian-bilaterian divergence. The deployment by corals of genes used by bacteria to compete with other bacteria reflects a mechanism of microbiome manipulation previously unknown in Metazoa but that may apply more generally.

Exploring Subtypes of Repetitive Behavior in Children with Autism Through Functional Analysis and Wearable Technology: a Pilot Biobehavioral Assessment.

Objectives: Prior research has established assessment methodologies, such as functional analysis to identify specific contexts in which restricted and repetitive behavior (RRB) occurs, and measures of heart rate variability (HRV) to index the level of autonomic arousal in individuals with autism spectrum disorder (ASD). Yet, a gap remains in integrating multiple assessment methodologies to examine the complex underlying mechanisms of RRB. This study piloted a multi-disciplinary approach to assess both the functional behavioral and neurophysiological factors that may underlie occurrences of RRB. The study (a) evaluated the effect of a modified functional analysis protocol on delineating functional subtypes of RRB and (b) explored the effect of using a wearable technology within a functional analysis on identifying the relationship between RRB and HRV. Method: A single-case alternating treatment design was used to randomly alternate noncontingent low-stimulation and high-stimulation conditions in a modified functional analysis protocol. Simultaneous measurement of RRB and HRV was obtained through direct behavioral observations and a wristband that collects blood volume pulse, respectively. Visual analysis of time series data was used to determine the functional subtypes of RRB, and nonparametric correlational analyses were conducted to determine the association between HRV and RRB. Results: Findings from a sample of six participants suggest preliminary effectiveness of the assessment protocol in identifying subtypes of RRB and a significant correlation between HRV and RRB. Conclusions: This study demonstrates the potential effect and usability of a wearable technology-aided biobehavioral approach to assess RRB and HRV in individuals with ASD.

Multiple opsins in a reef-building coral, Acropora millepora.

Opsins, light-sensitive G protein-coupled receptors, have been identified in corals but their properties are largely unknown. Here, we identified six opsin genes (acropsins 1-6) from a coral species Acropora millepora, including three novel opsins (acropsins 4-6), and successfully characterized the properties of four out of the six acropsins. Acropsins 1 and 6 exhibited light-dependent cAMP increases in cultured cells, suggesting that the acropsins could light-dependently activate Gs-type G protein like the box jellyfish opsin from the same opsin group. Spectral sensitivity curves having the maximum sensitivities at ~ 472 nm and ~ 476 nm were estimated for acropsins 1 and 6, respectively, based on the light wavelength-dependent cAMP increases in these opsins-expressing cells (heterologous action spectroscopy). Acropsin 2 belonging to the same group as acropsins 1 and 6 did not induce light-dependent cAMP or Ca2+ changes. We then successfully estimated the acropsin 2 spectral sensitivity curve having its maximum value at ~ 471 nm with its chimera mutant which possessed the third cytoplasmic loop of the Gs-coupled jellyfish opsin. Acropsin 4 categorized as another group light-dependently induced intracellular Ca2+ increases but not cAMP changes. Our results uncovered that the Acropora coral possesses multiple opsins coupling two distinct cascades, cyclic nucleotide and Ca2+signaling light-dependently.

Adult-Onset Myopathy with Constitutive Activation of Akt following the Loss of hnRNP-U.

Skeletal muscle has the remarkable ability to modulate its mass in response to changes in nutritional input, functional utilization, systemic disease, and age. This is achieved by the coordination of transcriptional and post-transcriptional networks and the signaling cascades balancing anabolic and catabolic processes with energy and nutrient availability. The extent to which alternative splicing regulates these signaling networks is uncertain. Here we investigate the role of the RNA-binding protein hnRNP-U on the expression and splicing of genes and the signaling processes regulating skeletal muscle hypertrophic growth. Muscle-specific Hnrnpu knockout (mKO) mice develop an adult-onset myopathy characterized by the selective atrophy of glycolytic muscle, the constitutive activation of Akt, increases in cellular and metabolic stress gene expression, and changes in the expression and splicing of metabolic and signal transduction genes. These findings link Hnrnpu with the balance between anabolic signaling, cellular and metabolic stress, and physiological growth.

The Effects of a Technology-Based Self-monitoring Intervention on On-Task, Disruptive, and Task-Completion Behaviors for Adolescents with Autism.

Individuals with autism spectrum disorders (ASD) often present with difficulty in sustaining engagement, attention, and have disruptive behavior in classroom settings. Without appropriate intervention, these challenging behaviors often persist and negatively impact educational outcomes. Self-monitoring is a well-supported evidence-based practice for addressing challenging behaviors and improving pro-social behaviors for individuals with ASD. Self-monitoring procedures utilizing a handheld computer-based technology is an unobtrusive and innovative way of implementing the intervention. A withdrawal design was employed to assess the effectiveness of a technologically-delivered self-monitoring intervention (I-Connect) in improving on-task and task completion behaviors and decreasing disruptive behavior with four adolescents with ASD. Results demonstrated improvements in on-task and task completion behaviors across all four participants and disruptive behavior improved for two participants.

Cognitive ability and math computation developmental relations with math problem solving: An integrated, multigroup approach.

Individual differences in Cattell-Horn-Carroll (CHC) cognitive abilities are related to individual differences in math problem solving. However, it is less clear whether cognitive abilities are associated with math problem solving directly or indirectly via math component skills and whether these relations differ across grade levels. We used multigroup structural equation models to examine direct and indirect CHC-based cognitive ability relations with math problem solving across six grade-level groups using the Kaufman Assessment Battery for Children, Second Edition and the Kaufman Tests of Educational Achievement, Second Edition co-normed standardization sample data (N = 2,117). After testing factorial invariance of the cognitive constructs across grade levels, we assessed whether the main findings were similar across higher-order and bifactor models. In the higher-order model, the Crystallized Ability, Visual Processing, and Short-Term Memory constucts had direct and indirect relations with math problem solving, whereas the Learning Efficiency and Retrieval Fluency constructs had only indirect relations with math problem solving via math computation. The integrated cognitive ability and math achievement relations were generally consistent across the CHC models of intelligence. In the higher-order model, the g factor operated indirectly on math computation and math problem solving, whereas in the bifactor model, the first-order G factor had direct relations with math computation and math problem solving. In both models, g/G was the most consistent and largest cognitive predictor of math skills. Last, the relation of math computation with math problem solving increased as grade level increased. Theoretical implications for math development and considerations for school psychologists are discussed. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Cognitive ability influences on written expression: Evidence for developmental and sex-based differences in school-age children.

Some studies have demonstrated that the Cattell-Horn-Carroll (CHC) cognitive abilities influence writing; however, little research has investigated whether CHC cognitive abilities influence writing the same way for males and females across grades. We used multiple group structural equation models to investigate whether CHC cognitive ability influences on written expression differed between grades or sex using the Kaufman Assessment Battery for Children, Second Edition and the Kaufman Tests of Educational Achievement, Second Edition co-normed standardization sample data (N=2117). After testing for consistent measurement of cognitive abilities across grades and sex, we tested whether the cognitive ability influences on written expression were moderated by grade level or sex. An important developmental shift was observed equally across sex groups: Learning Efficiency (Gl) influences decreased whereas Crystallized Ability (Gc) influences increased after fourth grade. Further, Short-Term Memory (Gsm) and Retrieval Fluency (Gr) influences on written expression depended on sex at grades 1-4, with larger Gr influences for females and larger Gsm influences for males. We internally replicated our main findings using two different cognitive explanatory models, adding further support for the developmental and sex-based differential cognitive ability influences on writing. Explanatory cognitive models of writing need to incorporate development, and possibly, sex to provide an expanded understanding of writing development and guard against potential generalizability issues characteristic of special population (i.e., male-female) studies.

Teacher-student relationship quality and academic achievement in elementary school: A longitudinal examination of gender differences.

Multiple group longitudinal cross-lagged panel models were implemented to understand the directional influences between teacher-student closeness and conflict and measured math and reading achievement across elementary grades and gender groups using the National Institute of Child Health and Human Development longitudinal sample (N=1133). Specifically, after testing multiple group longitudinal measurement invariance to ensure consistent measurement across genders and time, and tests of equivalence of the latent parameters, we were interested in whether longitudinal changes in teacher-rated closeness and conflict explained longitudinal changes in achievement, and vice versa, and whether those longitudinal influences varied by gender. Latent teacher-student closeness decreased for both genders over time (Cohen's d=-0.15 to -0.32), but latent conflict increased for males (Cohen's d=0.16). There was also increased heterogeneity in teacher-student relationship quality for males relative to females. Math and reading achievement had medium reciprocal effects (ß=0.12 to 0.23), and previous math achievement had small to medium effects on subsequent teacher-student closeness (ß=0.08 to 0.11) and conflict (ß=-0.07 to -0.09). Teacher-student conflict and closeness did not influence subsequent levels of math or reading achievement once previous levels were controlled. Further, these influences were consistent across gender groups despite latent differences in teacher-student closeness and conflict with teachers reporting closer relationships with female students and more conflictual relationships with male students.

Absorption Characteristics of Vertebrate Non-Visual Opsin, Opn3.

Most animals possess multiple opsins which sense light for visual and non-visual functions. Here, we show spectral characteristics of non-visual opsins, vertebrate Opn3s, which are widely distributed among vertebrates. We successfully expressed zebrafish Opn3 in mammalian cultured cells and measured its absorption spectrum spectroscopically. When incubated with 11-cis retinal, zebrafish Opn3 formed a blue-sensitive photopigment with an absorption maximum around 465 nm. The Opn3 converts to an all-trans retinal-bearing photoproduct with an absorption spectrum similar to the dark state following brief blue-light irradiation. The photoproduct experienced a remarkable blue-shift, with changes in position of the isosbestic point, during further irradiation. We then used a cAMP-dependent luciferase reporter assay to investigate light-dependent cAMP responses in cultured cells expressing zebrafish, pufferfish, anole and chicken Opn3. The wild type opsins did not produce responses, but cells expressing chimera mutants (WT Opn3s in which the third intracellular loops were replaced with the third intracellular loop of a Gs-coupled jellyfish opsin) displayed light-dependent changes in cAMP. The results suggest that Opn3 is capable of activating G protein(s) in a light-dependent manner. Finally, we used this assay to measure the relative wavelength-dependent response of cells expressing Opn3 chimeras to multiple quantally-matched stimuli. The inferred spectral sensitivity curve of zebrafish Opn3 accurately matched the measured absorption spectrum. We were unable to estimate the spectral sensitivity curve of mouse or anole Opn3, but, like zebrafish Opn3, the chicken and pufferfish Opn3-JiL3 chimeras also formed blue-sensitive pigments. These findings suggest that vertebrate Opn3s may form blue-sensitive G protein-coupled pigments. Further, we suggest that the method described here, combining a cAMP-dependent luciferase reporter assay with chimeric opsins possessing the third intracellular loop of jellyfish opsin, is a versatile approach for estimating absorption spectra of opsins with unknown signaling cascades or for which absorption spectra are difficult to obtain.

Reversal of ocean acidification enhances net coral reef calcification.

Approximately one-quarter of the anthropogenic carbon dioxide released into the atmosphere each year is absorbed by the global oceans, causing measurable declines in surface ocean pH, carbonate ion concentration ([CO3(2-)]), and saturation state of carbonate minerals (Ω). This process, referred to as ocean acidification, represents a major threat to marine ecosystems, in particular marine calcifiers such as oysters, crabs, and corals. Laboratory and field studies have shown that calcification rates of many organisms decrease with declining pH, [CO3(2-)], and Ω. Coral reefs are widely regarded as one of the most vulnerable marine ecosystems to ocean acidification, in part because the very architecture of the ecosystem is reliant on carbonate-secreting organisms. Acidification-induced reductions in calcification are projected to shift coral reefs from a state of net accretion to one of net dissolution this century. While retrospective studies show large-scale declines in coral, and community, calcification over recent decades, determining the contribution of ocean acidification to these changes is difficult, if not impossible, owing to the confounding effects of other environmental factors such as temperature. Here we quantify the net calcification response of a coral reef flat to alkalinity enrichment, and show that, when ocean chemistry is restored closer to pre-industrial conditions, net community calcification increases. In providing results from the first seawater chemistry manipulation experiment of a natural coral reef community, we provide evidence that net community calcification is depressed compared with values expected for pre-industrial conditions, indicating that ocean acidification may already be impairing coral reef growth.

Functional conservation of the apoptotic machinery from coral to man: the diverse and complex Bcl-2 and caspase repertoires of Acropora millepora.

BACKGROUND: Apoptotic cell death is a defining and ubiquitous characteristic of metazoans, but its evolutionary origins are unclear. Although Caenorhabditis and Drosophila played key roles in establishing the molecular bases of apoptosis, it is now clear that cell death pathways of these animals do not reflect ancestral characteristics. Conversely, recent work suggests that the apoptotic networks of cnidarians may be complex and vertebrate-like, hence characterization of the apoptotic complement of representatives of the basal cnidarian class Anthozoa will help us to understand the evolution of the vertebrate apoptotic network. RESULTS: We describe the Bcl-2 and caspase protein repertoires of the coral Acropora millepora, making use of the comprehensive transcriptomic data available for this species. Molecular phylogenetics indicates that some Acropora proteins are orthologs of specific mammalian pro-apoptotic Bcl-2 family members, but the relationships of other Bcl-2 and caspases are unclear. The pro- or anti-apoptotic activities of coral Bcl-2 proteins were investigated by expression in mammalian cells, and the results imply functional conservation of the effector/anti-apoptotic machinery despite limited sequence conservation in the anti-apoptotic Bcl-2 proteins. A novel caspase type ("Caspase-X"), containing both inactive and active caspase domains, was identified in Acropora and appears to be restricted to corals. When expressed in mammalian cells, full-length caspase-X caused loss of viability, and a truncated version containing only the active domain was more effective in inducing cell death, suggesting that the inactive domain might modulate activity in the full-length protein. Structure prediction suggests that the active and inactive caspase domains in caspase-X are likely to interact, resulting in a structure resembling that of the active domain in procaspase-8 and the inactive caspase domain in the mammalian c-FLIP anti-apoptotic factor. CONCLUSIONS: The data presented here confirm that many of the basic mechanisms involved in both the intrinsic and extrinsic apoptotic pathways were in place in the common ancestor of cnidarians and bilaterians. With the identification of most or all of the repertoires of coral Bcl-2 and caspases, our results not only provide new perspectives on the evolution of apoptotic pathways, but also a framework for future experimental studies towards a complete understanding of coral bleaching mechanisms, in which apoptotic cell death might be involved.

Technology delivered self-monitoring application to promote successful inclusion of an elementary student with autism.

The ever-increasing prevalence of children diagnosed with autism spectrum disorder (ASD) is paralleled in public educational settings, including general education classrooms. Challenges with social/behavioral functioning, including limited self-management and behavior inhibition, can lead to off-task and disruptive behaviors that interfere with acquisition of academic and social skills. Without effective and efficient interventions, opportunities to participate in inclusive settings will likely be reduced. Self-monitoring (SM) is an intervention with strong evidence for increasing prosocial behaviors and decreasing challenging behaviors for students with ASD in educational settings, although the cuing mechanisms (e.g., timers, stopwatch) and tracking materials (e.g., paper, pencil) can be cumbersome and obtrusive. I-Connect is an SM application that allows for customizable prompts, recording, and data monitoring. The purpose of this study was to evaluate, utilizing an ABAB design, the functional relationship between implementation of I-Connect SM intervention and increases in on-task behavior with concurrent decreases in disruptive behavior for an elementary student with ASD in a general education classroom. Results indicate an immediate increase in on-task behavior as well as a decrease in disruptive behaviors with each introduction of I-Connect. Implications for practice and future research are discussed.

Use of a Self-monitoring Application to Reduce Stereotypic Behavior in Adolescents with Autism: A Preliminary Investigation of I-Connect.

Many students with autism engage in a variety of complex stereotypic behaviors, impacting task completion and interfering with social opportunities. Self-monitoring is an intervention with empirical support for individuals with ASD to increase behavioral repertoires and decrease behaviors that are incompatible with successful outcomes. However, there is limited evidence for its utility for decreasing stereotypy, particularly for adolescents in school settings. This study evaluated the functional relationship between I-Connect, a technology-delivered self-monitoring program, and decreases in the level of stereotypy for two students with ASD in the school setting utilizing a withdrawal design with an embedded multiple baseline across participants. Both students demonstrated a marked decrease in stereotypy with the introduction of the self-monitoring application. Results and implications for practice and future research will be discussed.

Accuracy of optic nerve sheath diameter measurement by emergency physicians using bedside ultrasound.

BACKGROUND: Ultrasound (US) measurement of the optic nerve sheath diameter (ONSD) has been utilized as an indirect assessment of intracranial pressure. It is usually performed by trained ultrasonographers. OBJECTIVES: To evaluate whether or not emergency physicians (EP) are capable of measuring the ONSD accurately by US. MATERIALS AND METHODS: A retrospective measurement of ONSD was conducted on computed tomography (CT) scans of the head or facial bones. These patients had undergone ocular US performed by EPs prior to CT scanning. The CT scan measurements of ONSD read by a board-certified radiologist were compared with that of the US read by a registered diagnostic medical sonographer. A difference in measurements of the ONSD ≥ 0.5 mm between the two modalities was considered as significant for this study. RESULTS: The ONSD measurements were performed with CT scan and compared to that of the US. Of the 61 patients studied, 36 (59%) were male and 25 (41%) were female. The average age was 56 ± 17 years. All but 4 patients had ONSD measurements that were between 5 and 6 mm [Corrected]. Discrepancy in measurements of the ONSD between US and CT for both groups fell within our predetermined value (0.5 mm) for the majority of cases. None of the measurements were above 6 mm. The intraclass correlation coefficient was 0.9 (95% confidence interval 0.8846-0.9303). CONCLUSION: Emergency physicians were capable of accurately measuring the ONSD using bedside US. Prospective studies with a larger sample size are recommended to validate these findings.

Implementation of a self-monitoring application to improve on-task behavior: A high school pilot study.

Technological innovations offer promise for improving intervention implementation in secondary, inclusive classrooms. A withdrawal design was employed with two high school students in order to assess the effectiveness of a technologically-delivered, self-monitoring intervention in improving on-task behavior in a science classroom. Two students ages 14 and 15 with diagnoses of specific learning disability (student 1) and attention deficit hyperactivity disorder (ADHD: student 2) were selected by case manager referral due to difficulties with on-task behavior despite long-term administration of psychostimulant medication. After baseline data were collected, both students were trained in the use of a self-monitoring application (I-Connect) delivered via a handheld tablet. On-task prompts were delivered at five min intervals in an ABAB withdrawal design. The intervention resulted in positive, stable improvements in the primary dependent variable of on-task behavior for both students and less clear improvement in the generalization variable of disruptive behavior.

Projected near-future levels of temperature and pCO2 reduce coral fertilization success.

Increases in atmospheric carbon dioxide (pCO2) are projected to contribute to a 1.1-6.4°C rise in global average surface temperatures and a 0.14-0.35 reduction in the average pH of the global surface ocean by 2100. If realized, these changes are expected to have negative consequences for reef-building corals including increased frequency and severity of coral bleaching and reduced rates of calcification and reef accretion. Much less is known regarding the independent and combined effects of temperature and pCO2 on critical early life history processes such as fertilization. Here we show that increases in temperature (+3°C) and pCO2 (+400 µatm) projected for this century negatively impact fertilization success of a common Indo-Pacific coral species, Acropora tenuis. While maximum fertilization did not differ among treatments, the sperm concentration required to obtain 50% of maximum fertilization increased 6- to 8- fold with the addition of a single factor (temperature or CO2) and nearly 50- fold when both factors interact. Our results indicate that near-future changes in temperature and pCO2 narrow the range of sperm concentrations that are capable of yielding high fertilization success in A. tenuis. Increased sperm limitation, in conjunction with adult population decline, may have severe consequences for coral reproductive success. Impaired sexual reproduction will further challenge corals by inhibiting population recovery and adaptation potential.

Evidence for multiple phototransduction pathways in a reef-building coral.

Photosensitive behaviors and circadian rhythms are well documented in reef-building corals and their larvae, but the mechanisms responsible for photoreception have not been described in these organisms. Here we report the cloning, immunolocalization, and partial biochemical characterization of three opsins and four G proteins expressed in planulae of the Caribbean elkhorn coral, Acropora palmata. All three opsins (acropsins 1-3) possess conserved seven-pass transmembrane structure, and localize to distinct regions of coral planulae. Acropsin 1 was localized in the larval endoderm, while acropsin 2 was localized in solitary cells of the ectoderm. These rod-like cells displayed a remarkably polarized distribution, concentrated in the aboral end. We also cloned four A. palmata G protein alpha subunits. Three were homologs of vertebrate Gi, Go, and Gq. The fourth is presumably a novel G protein, which displays only 40% identity with the nearest known G protein, and we termed it Gc for "cnidarian". We show that Gc and Gq can be activated by acropsins in a light-dependent manner in vitro. This indicates that at least acropsins 1 and 3 can form functional photoreceptors and potentially may play a role in color preference during settlement, vertical positioning and other light-guided behaviors observed in coral larvae.

Long-wavelength photosensitivity in coral planula larvae.

Light influences the swimming behavior and settlement of the planktonic planula larvae of coral, but little is known regarding the photosensory biology of coral at this or any life-history stage. Here we used changes in the electrical activity of coral planula tissue upon light flashes to investigate the photosensitivity of the larvae. Recordings were made from five species: two whose larvae are brooded and contain algal symbionts (Porites astreoides and Agaricia agaricites), and three whose larvae are spawned and lack algal symbionts (Acropora cervicornis, Acropora palmata,and Montastrea faveolata). Photosensitivity originated from the coral larva rather than from, or in addition to, its algal symbionts as species with and without symbionts displayed similar tissue-level electrical responses to light. All species exhibited as much (or more) sensitivity to red stimuli as to blue/green stimuli, which is consistent with a role for long-wavelength visible light in the preference for substrata observed during settlement and in facilitating vertical positioning of larvae in the water column.

Ocean acidification compromises recruitment success of the threatened Caribbean coral Acropora palmata.

Ocean acidification (OA) refers to the ongoing decline in oceanic pH resulting from the uptake of atmospheric CO(2). Mounting experimental evidence suggests that OA will have negative consequences for a variety of marine organisms. Whereas the effect of OA on the calcification of adult reef corals is increasingly well documented, effects on early life history stages are largely unknown. Coral recruitment, which necessitates successful fertilization, larval settlement, and postsettlement growth and survivorship, is critical to the persistence and resilience of coral reefs. To determine whether OA threatens successful sexual recruitment of reef-building corals, we tested fertilization, settlement, and postsettlement growth of Acropora palmata at pCO(2) levels that represent average ambient conditions during coral spawning (∼400 µatm) and the range of pCO(2) increases that are expected to occur in this century [∼560 µatm (mid-CO(2)) and ∼800 µatm (high-CO(2))]. Fertilization, settlement, and growth were all negatively impacted by increasing pCO(2), and impairment of fertilization was exacerbated at lower sperm concentrations. The cumulative impact of OA on fertilization and settlement success is an estimated 52% and 73% reduction in the number of larval settlers on the reef under pCO(2) conditions projected for the middle and the end of this century, respectively. Additional declines of 39% (mid-CO(2)) and 50% (high-CO(2)) were observed in postsettlement linear extension rates relative to controls. These results suggest that OA has the potential to impact multiple, sequential early life history stages, thereby severely compromising sexual recruitment and the ability of coral reefs to recover from disturbance.

Long-term use of topical tacrolimus (FK506) in high-risk penetrating keratoplasty.

PURPOSE: To evaluate the long-term efficacy and side effects of off-label topical tacrolimus 0.03% ointment (Protopic; Fujisawa Health, Deerfield, IL) as a sole second-line immunosuppressive agent in the management of high-risk corneal grafts. METHODS: Four consecutive patients underwent high-risk penetrating keratoplasty (4 grafts) with a prior diagnosis of corneal scar secondary to herpetic keratitis, keratoconus, acanthamoeba keratitis, and Fuchs endothelial dystrophy, respectively. All 4 patients developed steroid-induced glaucoma and failed traditional immunosuppressant therapy. Patients were started on topical tacrolimus ointment 0.03%, twice daily, which was tapered to the lowest possible therapeutic dose that maintained its antirejection efficacy. Patients were monitored for adverse treatment effects. The mean follow-up was 33 months (range, 26-48 months), and the mean treatment duration was 22.6 months (range, 13-32 months). RESULTS: All 4 high-risk corneal transplant patients experienced episodes of acute rejection that was successfully reversed with topical tacrolimus treatment. During tacrolimus treatment, there were no further episodes of graft rejection and no incidents of herpes simplex virus infection or reactivation, with the longest follow-up being 4 years. Two patients have been successfully tapered off tacrolimus, and 2 patients are currently on once-daily dosing. No adverse effects were observed. CONCLUSIONS: Topical tacrolimus 0.03% ointment seems to be a promising second-line immunosuppressant in management of high-risk grafts.