Genome-wide study of gene-by-sex interactions identifies risks for cleft palate.

Structural birth defects affect 3-4% of all live births and, depending on the type, tend to manifest in a sex-biased manner. Orofacial clefts (OFCs) are the most common craniofacial structural birth defects and are often divided into cleft lip with or without cleft palate (CL/P) and cleft palate only (CP). Previous studies have found sex-specific risks for CL/P, but these risks have yet to be evaluated in CP. CL/P is more common in males and CP is more frequently observed in females, so we hypothesized there would also be sex-specific differences for CP. Using a trio-based cohort, we performed sex-stratified genome-wide association studies (GWAS) based on proband sex followed by a genome-wide gene-by-sex (GxS) interaction testing. There were 13 loci significant for GxS interactions, with the top finding in LTBP1 (RR=3.37 [2.04 - 5.56], p=1.93x10 -6 ). LTBP1 plays a role in regulating TGF-B bioavailability, and knockdown in both mice and zebrafish lead to craniofacial anomalies. Further, there is evidence for differential expression of LTBP1 between males and females in both mice and humans. Therefore, we tested the association between the imputed genetically regulated gene expression of genes with significant GxS interactions and the CP phenotype. We found significant association for LTBP1 in cell cultured fibroblasts in female probands (p=0.0013) but not in males. Taken altogether, we show there are sex-specific risks for CP that are otherwise undetectable in a combined sex cohort, and LTBP1 is a candidate risk gene, particularly in females.

The heterogeneous genetic architectures of orofacial clefts.

Orofacial clefts (OFCs) are common, affecting 1:1000 live births. OFCs occur across a phenotypic spectrum - including cleft lip (CL), cleft lip and palate (CLP), or cleft palate (CP) - and can be further subdivided based on laterality, severity, or specific structures affected. Herein we review what is known about the genetic architecture underlying each of these subtypes, considering both shared and subtype-specific risks. While there are more known genetic similarities between CL and CLP than CP, recent research supports both shared and subtype-specific genetic risk factors within and between phenotypic classifications of OFCs. Larger sample sizes and deeper phenotyping data will be of increasing importance for the discovery of novel genetic risk factors for OFCs and various subtypes going forward.

Utilizing Electromyographic Video Games Controllers to Improve Outcomes for Prosthesis Users.

A study was developed for a limb-different accessible video game controller that utilizes an electromyographic sensor to control gameplay actions. Data was collected from 50 college-aged student participants. This biofeedback-based serious game trains users in a virtual capacity, through the visualization of muscle contraction, via the movement of the video game character. The training platform has been developed to accompany the corresponding electromyographic actuated prosthetic arm device, leveraging the same control scheme to enable the translation of hand gesture states. This study evaluated the controller, user interface, and gameplay to identify training improvement outcomes and user satisfaction. Study participants were divided into two cohorts that differed in their intervention between the pre-test and post-test challenge course. Cohort one had a free play environment that encouraged learning through algorithmically generated track patterns and the use of powerups. In contrast, cohort two repeated the challenge mode, which was made up of a course of rings to jump through and focused on targeted muscle discretization via character jump heights correlated to muscle output. Data were collected to develop and validate training methods and identify overall game satisfaction and usability. The results of this study indicated an increase in the user's ability to be successful based on time on task with the intervention. The study also evaluated the usability and participant experience with the intervention.

Trio-based GWAS identifies novel associations and subtype-specific risk factors for cleft palate.

Cleft palate (CP) is one of the most common craniofacial birth defects; however, there are relatively few established genetic risk factors associated with its occurrence despite high heritability. Historically, CP has been studied as a single phenotype, although it manifests across a spectrum of defects involving the hard and/or soft palate. We performed a genome-wide association study using transmission disequilibrium tests of 435 case-parent trios to evaluate broad risks for any cleft palate (ACP) (n = 435), and subtype-specific risks for any cleft soft palate (CSP), (n = 259) and any cleft hard palate (CHP) (n = 125). We identified a single genome-wide significant locus at 9q33.3 (lead SNP rs7035976, p = 4.24 × 10-8) associated with CHP. One gene at this locus, angiopoietin-like 2 (ANGPTL2), plays a role in osteoblast differentiation. It is expressed both in craniofacial tissue of human embryos and developing mouse palatal shelves. We found 19 additional loci reaching suggestive significance (p < 5 × 10-6), of which only one overlapped between groups (chromosome 17q24.2, ACP and CSP). Odds ratios for the 20 loci were most similar across all 3 groups for SNPs associated with the ACP group, but more distinct when comparing SNPs associated with either subtype. We also found nominal evidence of replication (p < 0.05) for 22 SNPs previously associated with orofacial clefts. Our study to evaluate CP risks in the context of its subtypes and we provide newly reported associations affecting the broad risk for CP as well as evidence of subtype-specific risks.

Integrative analysis of transcriptome dynamics during human craniofacial development identifies candidate disease genes.

Craniofacial disorders arise in early pregnancy and are one of the most common congenital defects. To fully understand how craniofacial disorders arise, it is essential to characterize gene expression during the patterning of the craniofacial region. To address this, we performed bulk and single-cell RNA-seq on human craniofacial tissue from 4-8 weeks post conception. Comparisons to dozens of other human tissues revealed 239 genes most strongly expressed during craniofacial development. Craniofacial-biased developmental enhancers were enriched +/- 400 kb surrounding these craniofacial-biased genes. Gene co-expression analysis revealed that regulatory hubs are enriched for known disease causing genes and are resistant to mutation in the normal healthy population. Combining transcriptomic and epigenomic data we identified 539 genes likely to contribute to craniofacial disorders. While most have not been previously implicated in craniofacial disorders, we demonstrate this set of genes has increased levels of de novo mutations in orofacial clefting patients warranting further study.

Multiplex analysis of cytokines in the cerebrospinal fluid of dogs after ischemic stroke reveals elevations in chemokines CXCL1 and MCP-1.

Introduction: Neuroinflammation that occurs in the brain after stroke has been shown to be important to disease pathogenesis and outcomes. The aim of this study was to evaluate a large number of pro- and anti-inflammatory cytokines in dogs with clinically-confirmed, naturally occurring stroke. Materials and methods: Fifteen dogs with a clinical diagnosis of ischemic stroke and ten healthy control dogs were included in the study. A multiplex immunoassay was utilized to evaluate cerebrospinal fluid for GM-CSF, IFN-γ, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, IP-10, CXCL1, MCP-1, and TNF-α. Results: Mean concentrations of CXCL1 (stroke-436 pg/ml, control-267 pg/ml, p = 0.01) and MCP-1 (stroke-196 pg/ml, control-66 pg/ml, p ≤ 0.0001) were significantly elevated in dogs with stroke when compared with control dogs. Location and type of infarct, duration of clinical signs, and use of anti-inflammatory medications were not associated with differences in cytokine concentration. Discussion: CXCL1 and MCP-1 may play a role in naturally occurring canine stroke and represent targets for future research.

Measurement of School Engagement in Elementary School Students: A Scoping Review.

IMPORTANCE: School engagement is the extent to which students commit to and participate in school activities, including internal thoughts, emotions, and observable behaviors. It is critical to children's academic outcomes and mental health. Occupational therapy practitioners support children at school to maintain mental well-being and meet their school outcomes. However, how occupational therapy practitioners should measure school engagement among elementary school students remains unclear. OBJECTIVE: To identify and characterize how elementary school students' school engagement is currently measured. DATA SOURCES: PsycINFO, Eric, CINAHL, and A+ Education databases. Two reviewers screened titles and abstracts, and one reviewer completed full-text screening and data extraction using Excel. STUDY SELECTION: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews guided this review. Studies published between 2015 and 2021 were included if full text was available, written in English, and used a measure designed for elementary school-age students. Studies were excluded if they used no school engagement measurement; used only infant, adolescent, or adult scales; were not available for review; and did not meet the inclusion criteria. FINDINGS: The review included 125 studies. A range of self-report, observational, teacher-report, and caregiver-report measures of school engagement were identified. Behavioral school engagement was most commonly measured. Included studies were primarily published in education and psychology fields, with none published in occupational therapy journals. CONCLUSIONS AND RELEVANCE: A range of school engagement measurements can be found in the literature, but no consensus exists on a validated school engagement measurement for occupational therapy practice. What This Article Adds: This review provides occupational therapy practitioners with a comprehensive understanding of (1) the importance of school engagement to mental health and (2) the range of behavioral, cognitive, and emotional engagement measures currently available for use with elementary school-age children, thereby enhancing the profession's knowledge and scope of practice in school engagement.

Community perceptions of vaccine advocacy for children under five in rural Guatemala.

Historically, partnerships with community leaders (e.g., religious leaders, teachers) have been critical to building vaccination confidence, but leaders may be increasingly vaccine hesitant. In rural Guatemala, the extent of vaccine hesitancy among community leaders is unclear, as are their perceptions of advocacy for childhood vaccines. We sought to: (i) compare Guatemalan religious leaders' and community leaders' attitudes toward childhood vaccines, (ii) describe leaders' experiences and comfort with vaccination advocacy, and (iii) describe community members' trust in them as vaccination advocates. In 2019, we surveyed religious leaders, other community leaders, and parents of children under five in rural Guatemala. We recorded participant demographic information and assessed participant vaccine hesitancy regarding childhood vaccines. We analyzed data descriptively and via adjusted regression modeling. Our sample included 50 religious leaders, 50 community leaders, and 150 community members (response rate: 99%); 14% of religious leaders and community leaders were vaccine hesitant, similar to community members (P = 0.71). In the prior year, 47% of leaders had spoken about vaccines in their formal role; 85% felt responsible to do so. Only 28% of parents trusted politicians "a lot" for vaccine advice, versus doctors (72%; P < 0.01), nurses (62%; P < 0.01), religious leaders (49%; P < 0.01), and teachers (48%; P < 0.01). In this study, religious leaders and community leaders were willing but incompletely engaged vaccination advocates. Most community members trusted doctors and nurses a lot for vaccination advice; half trusted teachers and religious leaders similarly. Public health officials in rural Guatemala can complement efforts by doctors and nurses through partnerships with teachers and religious leaders to increase vaccination confidence and delivery.

Trio-based GWAS identifies novel associations and subtype-specific risk factors for cleft palate.

Orofacial clefts (OFCs) are the most common craniofacial birth defects and are often categorized into two etiologically distinct groups: cleft lip with or without cleft palate (CL/P) and isolated cleft palate (CP). CP is highly heritable, but there are still relatively few established genetic risk factors associated with its occurrence compared to CL/P. Historically, CP has been studied as a single phenotype despite manifesting across a spectrum of defects involving the hard and/or soft palate. We performed GWAS using transmission disequilibrium tests using 435 case-parent trios to evaluate broad risks for any cleft palate (ACP, n=435), as well as subtype-specific risks for any cleft soft palate (CSP, n=259) and any cleft hard palate (CHP, n=125). We identified a single genome-wide significant locus at 9q33.3 (lead SNP rs7035976, p=4.24×10 -8 ) associated with CHP. One gene at this locus, angiopoietin-like 2 ( ANGPTL2 ), plays a role in osteoblast differentiation. It is expressed in craniofacial tissue of human embryos, as well as in the developing mouse palatal shelves. We found 19 additional loci reaching suggestive significance (p<5×10 -6 ), of which only one overlapped between groups (chromosome 17q24.2, ACP and CSP). Odds ratios (ORs) for each of the 20 loci were most similar across all three groups for SNPs associated with the ACP group, but more distinct when comparing SNPs associated with either the CSP or CHP groups. We also found nominal evidence of replication (p<0.05) for 22 SNPs previously associated with cleft palate (including CL/P). Interestingly, most SNPs associated with CL/P cases were found to convey the opposite effect in those replicated in our dataset for CP only. Ours is the first study to evaluate CP risks in the context of its subtypes and we provide newly reported associations affecting the broad risk for CP as well as evidence of subtype-specific risks.

Measurement of Self-regulation in Preschool and Elementary Children: A Scoping Review.

AIMS: This scoping review sought to identify and characterize measurement of self-regulation in preschool and elementary aged children. METHODS: The Preferred Reporting Items for Systematic Reviews and Meta-Analyses-Scoping Review (ScR) guidelines were applied. Databases from the fields of allied health, education, medicine, and social sciences were searched including: CINAHL, Education Database (ProQuest), Education Research Complete, EMBASE, ERIC, iNFORMIT Combined, Medline, PsychINFO, Social Sciences (ProQuest), Teacher Reference Center, and Web of Science. Articles published between 2015 and 2020 were included. Dual review was utilized at all stages and a third reviewer resolved any conflicts. RESULTS: Sixty-seven studies were included in this review. A range of observational, self-report, teacher report, caregiver report, and observational measures of self-regulation were identified. Included studies were primarily published in education and psychology disciplines, with no studies by occupational therapists identified. CONCLUSIONS: Although a range of measures were identified in this scoping review, the results highlight the lack of consensus regarding self-regulation measurement that occupational therapists use to design and implement therapy programs to address child emotional and behavioral needs.

Safety and Efficacy of Medical Cannabis in Autism Spectrum Disorder Compared with Commonly Used Medications.

Objective: The objective of this study was to evaluate the safety and efficacy of medications commonly used in autism spectrum disorder (ASD) and compare this to what current research has shown regarding medical cannabis use in this population. Methods: Searches were performed to collect information surrounding currently used medications and their safety and efficacy profiles, biologic plausibility of cannabis use for symptoms of ASD, and studies detailing cannabis' safety and efficacy profile for use in the ASD population. Results were used to compare medications to cannabis as a proposed treatment. Results: The heterogeneity of ASD produces great difficulties in finding appropriate treatment, leading to many medication changes or treatment trials throughout a patient's life. Commonly prescribed medications display varying levels of efficacy, safety, and tolerability between patients and symptoms targeted. Some of the most common side effects cited are also considered the most troubling symptoms associated with ASD; aggression, anxiety, irritability, and a negative effect on cognition, leading many patients to discontinue use as the side effects outweigh benefits. Recent case reports and retrospective studies have displayed the potential efficacy, safety, and tolerability of cannabidiol (CBD)-rich medical cannabis use for treating both core symptoms of ASD and many comorbid symptoms such as irritability and sleep problems. Studies have also identified circulating endocannabinoids as a possible biomarker for ASD, providing another possible method of diagnosis. Conclusions: Currently, there are no approved medications for the core symptoms of ASD and only two medications Food and Drug Administration approved for associated irritability. Prescribed medications for symptoms associated with ASD display varying levels of efficacy, safety, and tolerability among the heterogeneous ASD population. At the time of this study there are no published placebo-controlled trials of medical cannabis for ASD and the observational studies have limitations. CBD-rich medical cannabis seems to be an effective, tolerable, and relatively safe option for many symptoms associated with ASD, however, the long-term safety is unknown at this time.

Identification of serum microRNAs with differential expression between dogs with splenic masses and healthy dogs with histologically normal spleens.

OBJECTIVE: To identify differential microRNA (miRNA) expression in dogs with splenic hemangiosarcoma, splenic hematoma, and histologically normal spleens. ANIMALS: Dogs with splenic hemangiosarcoma (n = 10), splenic hematoma (n = 5), and histologically normal spleens (n = 5). PROCEDURES: Splenic tissue and serum samples were collected from dogs with splenic masses (ie, hemangiosarcoma or hematoma samples) and healthy control dogs (ie, control samples), and total RNA was extracted. Reverse transcription quantitative real-time PCR was performed with 28 miRNAs associated with hemangiosarcoma, angiosarcoma, or associated genes. Differential expression analysis was performed. RESULTS: Control tissue and serum samples had similar miRNA expression patterns, and hemangiosarcoma tissue and serum samples did not. Hemangiosarcoma serum samples had higher expression than hemangiosarcoma tissue for 13 miRNAs and lower expression for 1 miRNA. Control tissue and hemangiosarcoma tissue had varying expressions for 12 miRNAs, with 10 more highly expressed in control samples and 2 more highly expressed in hemangiosarcoma samples. Five miRNAs (miR-214-3p, miR-452, miR-494-3p, miR-497-5p, miR-543) had significantly different expression in serum between dogs with splenic masses (ie, hemangiosarcoma or hematoma) and serum of dogs with histologically normal spleens, with higher expression in the serum of dogs with splenic masses for all 5 miRNAs. CONCLUSIONS AND CLINICAL RELEVANCE: 5 circulating miRNAs were identified that distinguished dogs with splenic hemangiosarcoma or hematoma from those with histologically normal spleens. These 5 miRNAs had higher expression in dogs with splenic masses, indicating upregulation of these circulating miRNAs occurs in these splenic disease states. These miRNAs may be useful as a noninvasive screening tool that uses serum to identify dogs with splenic masses.

Innovative Virtual Role Play Simulations for Managing Substance Use Conversations: Pilot Study Results and Relevance During and After COVID-19.

BACKGROUND: Substance use places a substantial burden on our communities, both economically and socially. In light of COVID-19, it is predicted that as many as 75,000 more people will die from alcohol and other substance use and suicide as a result of isolation, new mental health concerns, and various other stressors related to the pandemic. Public awareness campaigns that aim to destigmatize substance use and help individuals have meaningful conversations with friends, coworkers, or family members to address substance use concerns are a timely and cost-effective means of augmenting existing behavioral health efforts related to substance use. These types of interventions can supplement the work being done by existing public health initiatives. OBJECTIVE: This pilot study examines the impact of the One Degree: Shift the Influence role play simulation, designed to teach family, friends, and coworkers to effectively manage problem-solving conversations with individuals that they are concerned about regarding substance use. METHODS: Participants recruited for this mixed methods study completed a presurvey, the simulation, and a postsurvey, and were sent a 6-week follow-up survey. The simulation involves practicing a role play conversation with a virtual human coded with emotions, a memory, and a personality. A virtual coach provides feedback in using evidence-based communication strategies such as motivational interviewing. RESULTS: A matched sample analysis of variance revealed significant increases at follow-up in composite attitudinal constructs of preparedness (P<.001) and self-efficacy (P=.01), including starting a conversation with someone regarding substance use, avoiding upsetting someone while bringing up concerns, focusing on observable facts, and problem solving. Qualitative data provided further evidence of the simulation's positive impact on the ability to have meaningful conversations about substance use. CONCLUSIONS: This study provides preliminary evidence that conversation-based simulations like One Degree: Shift the Influence that use role play practice can teach individuals to use evidence-based communication strategies and can cost-effectively reach geographically dispersed populations to support public health initiatives for primary prevention.

Peri-ictal magnetic resonance imaging characteristics in dogs with suspected idiopathic epilepsy.

BACKGROUND: The pathophysiology of changes in magnetic resonance imaging (MRI) detected after a seizure is not fully understood. OBJECTIVE: To characterize and describe seizure-induced changes detected by MRI. ANIMALS: Eighty-one client-owned dogs diagnosed with idiopathic epilepsy. METHODS: Data collected retrospectively from medical records and included anatomical areas affected, T1-, T2-weighted and T2-FLAIR (fluid-attenuated inversion recovery) appearance, whether changes were unilateral or bilateral, symmetry, contrast enhancement, mass effect, and, gray and white matter distribution. Diffusion- and perfusion weighted maps were evaluated, if available. RESULTS: Seizure-induced changes were T2-hyperintense with no suppression of signal on FLAIR. Lesions were T1-isointense (55/81) or hypointense (26/81), local mass effect (23/81) and contrast enhancement (12/81). The majority of changes were bilateral (71/81) and symmetrical (69/71). The most common areas affected were the hippocampus (39/81) cingulate gyrus (33/81), hippocampus and piriform lobes (32/81). Distribution analysis suggested concurrence between cingulate gyrus and pulvinar thalamic nuclei, the cingulate gyrus and parahippocampal gyrus, hippocampus and piriform lobe, and, hippocampus and parahippocampal gyrus. Diffusion (DWI) characteristics were a mixed-pattern of restricted, facilitated, and normal diffusion. Perfusion (PWI) showed either hypoperfusion (6/9) or hyperperfusion (3/9). CONCLUSIONS AND CLINICAL IMPORTANCE: More areas, than previously reported, have been identified that could incur seizure-induced changes. Similar to human literature, DWI and PWI changes have been identified that could reflect the underlying metabolic and vascular changes.

Canine cognitive dysfunction patients have reduced total hippocampal volume compared with aging control dogs: A comparative magnetic resonance imaging study.

Background: Hippocampal atrophy is a key pathologic and magnetic resonance imaging (MRI) feature of human Alzheimer's disease (AD). Hippocampal atrophy has not been documented via MRI in canine cognitive dysfunction (CCD), which is considered as the dog model of human AD. Aim: The purpose of this retrospective comparative volumetric MRI study was to compare total hippocampal volumes between successfully aging (control) dogs and dogs diagnosed with CCD. Methods: Mimics® software was used to derive total hippocampal volumes and total brain volumes from the MRI studies of 42 aging dogs (≥ 9 years): 16 dogs diagnosed with CCD and 26 successfully aging controls. Hippocampal volumes were normalized to total brain volume and these values were compared between groups using Mann-Whitney U tests. Results: Total hippocampal volume normalized to total brain volume was significantly less for CCD patients compared with control dogs (p = 0.04). Conclusion: The results of this study suggest that - similar to human AD - hippocampal atrophy is a pathological feature of CCD. This finding has potential importance for both investigating disease mechanisms related to dementia as well as future hippocampal-targeted therapies.

Adapting and piloting a vaccine hesitancy questionnaire in rural Guatemala.

INTRODUCTION: We sought to (i) adapt a Spanish-language vaccine hesitancy (VH) tool to rural Guatemala, (ii) pilot the tool with 150 parents of children ≤ 5 years, and (iii) measure if parent scores associated with child under-vaccination. METHODS: We used implementation science to develop the adapted Guatemalan Vaccine Attitudes (GuaVA) tool, piloting it with 150 parents of children ≤ 5 years, and performing descriptive and adjusted regression analyses. RESULTS: Of 150 parents (response rate 99%), 55% (n = 83) of parents expressed a degree of VH. Children of parents with highly hesitant scores (n = 22) had 2.5 times the odds (OR 2.5; 95% CI: 1.2, 5.4) of being undervaccinated at 19 months, referent children of non-hesitant parents (n = 67). CONCLUSIONS: Vaccine hesitancy may be more prevalent in rural Guatemala than suspected. Implementation science facilitated the adaptation of a VH tool to rural Guatemala and may assist investigators in other settings.

Feasibility of Non-Invasive Vagus Nerve Stimulation (gammaCore VET™) for the Treatment of Refractory Seizure Activity in Dogs.

Idiopathic epilepsy is the most common chronic neurologic condition in dogs. Approximately 20-30% of those dogs are refractory to standard medical therapy and commonly experience side effects from antiepileptic drugs. Non-invasive vagus nerve stimulation (nVNS) has been frequently used in human medicine as an adjunct seizure therapy with low incidence of adverse events. Canine studies are limited to invasive surgical implants with no non-invasive evaluations currently published. We investigated the feasibility and efficacy of nVNS (gammaCore VET) as an adjunct treatment for refractory epilepsy in dogs. In total, 14 client-owned dogs completed the trial of either 8- or 16-week treatment periods during which they received 90-120 s stimulation three times per day in the region of the left cervical vagus nerve. Owners recorded seizure type (focal or generalized) and frequency as well as any adverse effects. Out of 14 dogs, nine achieved a reduction in seizure frequency and four were considered responders with a 50% or greater reduction in seizures from baseline to the final treatment period. However, there was no statistically significant difference in overall seizure frequency (p = 0.53) or percent change in seizure frequency between groups (p = 0.75). Adverse effects occurred in 25% of dogs originally enrolled, with reports of a hoarse bark and limb trembling, lethargy, behavioral changes, and an increase in seizure frequency. Non-invasive VNS was found to be safe and easy to administer with mild adverse events. It is considered a feasible treatment option as an adjunct therapy in refractory seizures and should be further investigated.

Hydroxylamine Complexes of Cytochrome c': Influence of Heme Iron Redox State on Kinetic and Spectroscopic Properties.

Hydroxylamine (NH2OH or HA) is a redox-active nitrogen oxide that occurs as a toxic intermediate in the oxidation of ammonium by nitrifying and methanotrophic bacteria. Within ammonium containing environments, HA is generated by ammonia monooxygenase (nitrifiers) or methane monooxygenase (methanotrophs). Subsequent oxidation of HA is catalyzed by heme proteins, including cytochromes P460 and multiheme hydroxylamine oxidoreductases, the former contributing to emissions of N2O, an ozone-depleting greenhouse gas. A heme-HA complex is also a proposed intermediate in the reduction of nitrite to ammonia by cytochrome c nitrite reductase. Despite the importance of heme-HA complexes within the biogeochemical nitrogen cycle, fundamental aspects of their coordination chemistry remain unknown, including the effect of the Fe redox state on heme-HA affinity, kinetics, and spectroscopy. Using stopped-flow UV-vis and resonance Raman spectroscopy, we investigated HA complexes of the L16G distal pocket variant of Alcaligenes xylosoxidans cytochrome c'-α (L16G AxCP-α), a pentacoordinate c-type cytochrome that we show binds HA in its Fe(III) (Kd ∼ 2.5 mM) and Fe(II) (Kd = 0.0345 mM) states. The ∼70-fold higher HA affinity of the Fe(II) state is due mostly to its lower koff value (0.0994 s-1 vs 11 s-1), whereas kon values for Fe(II) (2880 M-1 s-1) and Fe(III) (4300 M-1 s-1) redox states are relatively similar. A comparison of the HA and imidazole affinities of L16G AxCP-α was also used to predict the influence of Fe redox state on HA binding to other proteins. Although HA complexes of L16G AxCP-α decompose via redox reactions, the lifetime of the Fe(II)HA complex was prolonged in the presence of excess reductant. Spectroscopic parameters determined for the Fe(II)HA complex include the N-O stretching vibration of the NH2OH ligand, ν(N-O) = 906 cm-1. Overall, the kinetic trends and spectroscopic benchmarks from this study provide a foundation for future investigations of heme-HA reaction mechanisms.

A Pilot Study on the Safety of a Novel Antioxidant Nanoparticle Delivery System and Its Indirect Effects on Cytokine Levels in Four Dogs.

Acute spinal cord injury consists of a primary, traumatic event followed by a cascade of secondary events resulting in ongoing cell damage and death. There is great interest in prevention of these secondary effects to reduce permanent long-term neurologic deficits. One such target includes reactive oxygen species released following injury, which can be enzymatically converted into less harmful molecules by superoxide dismutase and catalase. Canine intervertebral disc herniation has been suggested as a naturally occurring model for acute spinal cord injury and its secondary effects in people. The aims of this study were to test the safety of a novel antioxidant delivery system in four healthy dogs and to indirectly test effect of delivery via cytokine measurement. All dogs experienced adverse events to some degree, with two experiencing adverse events considered to be severe. The clinical signs, including combinations of bradycardia, hypotension, hypersalivation, pale gums, and involuntary urination, were consistent with complement activation-related pseudoallergy (CARPA). CARPA is a well-known phenomenon that has been reported to occur with nanoparticle-based drug delivery, among other documented causes. Two dogs also had mild to moderate changes in their blood cell count and chemistry, including elevated alanine transferase, and thrombocytopenia, which both returned to normal by day 7 post-administration. Cytokine levels trended downwards over the first 3 days, but many were elevated at measurement on day 7. Intradermal testing suggested catalase as a potential cause for reactions. No long-term clinical signs were observed, and necropsy results revealed no concerning pathology. Additional evaluation of this product, including further characterization of reactions to catalase containing components, dose-escalation, and desensitization should be performed before evaluation in clinically affected dogs.