Simultaneous determination of steroid hormones and pharmaceuticals in killer whale (Orcinus orca) faecal samples by liquid chromatography tandem mass spectrometry.

We describe a non-invasive method for profiling selected hormones, pharmaceuticals and personal care products (PPCPs) in killer whales (Orcinus orca) based on analysis of faecal samples by liquid chromatography tandem mass spectrometry (LC-MS/MS). The method targets 21 compounds of interest including glucocorticoids, mineralocorticoids, androgens, estrogens, progestogens, selective serotonin uptake inhibitors and an antibacterial/antifungal agent. This method is suitable for routine simultaneous determination of target compounds in killer whale faecal samples as well as validation of immunoassays for the detection and measurement of steroid hormones in faeces. The optimized method involves extraction of freeze-dried faecal material with reagent alcohol and water followed by isolation of the analytes using solid phase extraction with hydrophilic-lipophilic balance cartridges and liquid-liquid extraction with methyl tertiary-butyl ether. Reconstituted extracts were analysed by LC-MS/MS using an electrospray ionization interface. Method limit of quantification ranged from 0.06 to 45.2 ng/g in freeze-dried faecal samples. Except for sertraline, triclosan and estradiol (which was not recovered at the lowest spiked concentration), average intra- and inter-day precisions were within 10%, and average recoveries were between 89.3% and 129.3%, for faecal samples spiked with 5.3, 26.7 or 133 ng/g of each analyte. The method was applied successfully to the analysis of hormones and PPCPs in whale faeces during which 17α-hydroxyprogesterone, a common intermediate in steroid biosynthesis that cross-reacts with precursors and sulphated conjugates in immunoassays, was identified and quantified in all samples.

Changing environmental conditions have altered the feeding ecology of two keystone Arctic marine predators.

Environmental change in the Arctic has impacted the composition and structure of marine food webs. Tracking feeding ecology changes of culturally-valued Arctic char (Salvelinus alpinus) and ringed seals (Pusa hispida) can provide an indication of the ecological significance of climate change in a vulnerable region. We characterized how changes in sea ice conditions, sea surface temperature (SST), and primary productivity affected the feeding ecology of these two keystone species over a 13- and 18-year period, respectively, in northern Labrador, Canada. Arctic char fed consistently on pelagic resources (δ13C) but shifted over time to feeding at a higher trophic level (δ15N) and on more marine/offshore resources (δ34S), which correlated with decreases in chlorophyll a concentration. A reduction in Arctic char condition factor and lipid content was associated with higher trophic position. Ringed seals also shifted to feeding at a higher trophic level, but on more pelagic resources, which was associated with lower SST and higher chlorophyll a concentrations. Years with abnormally high SSTs and reduced sea ice concentrations resulted in large isotopic niche sizes for both species, suggesting abrupt change can result in more variable feeding. Changes in abundance and distribution of species long valued by the Inuit of Labrador could diminish food security.

Sediment Spatial Distribution and Quality Assessment of Metals in Chinook Salmon and Resident Killer Whale Marine Habitat in British Columbia, Canada.

At-risk resident killer whale (Orcinus orca) populations of the northeastern Pacific, Canada, and their main prey, Chinook Salmon (Oncorhynchus tshawytscha), are exposed to a variety of contaminants including chemical elements from both natural and anthropogenic sources, which may be constraining their recovery. Concentrations of 36 chemical elements in subtidal surface sediments (1-435 m depth) collected from 98 sites along the British Columbia coast were used to characterize coast-wide patterns, and a subset of metals (mercury (Hg), cadmium (Cd), arsenic (As), nickel (Ni), copper (Cu), and lead (Pb)) were selected to assess Chinook Salmon and resident killer whale marine habitat quality. Principal component analysis (PCA) showed a dominance of Hg, antimony (Sb), Pb, Cu, and zinc (Zn) for Prince Rupert Harbour, Victoria Harbour, and Burrard Inlet, suggesting local sources. Based on the PCA, geochemical properties such as total organic carbon (TOC), acid volatile sulfide (AVS), and pH explained the spatial distribution of all elements in sediment (p < 0.001). Mercury, Cd, As, Ni, Cu, and Pb hotspots were identified along the coast of Vancouver Island, the central and north coast, in the Strait of Georgia, and Haida Gwaii. Bischof Island of Haida Gwaii and Ardmillan Bay on the central coast were most contaminated and enriched by Cd, determined by geoaccumulation index (Igeo) and enrichment factor (EF), respectively. Marine habitat quality was assessed by comparing metal concentrations to Canadian Sediment Quality Guidelines (SQGs). Chinook Salmon populations may be indirectly affected by metal toxicity (As > Cd and Cu > Ni > Hg > Pb) to lower trophic level prey species. Toxicity related impacts to benthic organisms as a result of exposure to elevated Cd and As concentrations in Northern Resident Killer Whale critical habitat and to Hg, Cd, As, Ni, Cu, and Pb concentrations in Southern Resident Killer Whale critical habitat may indirectly pose a threat to resident killer whale populations, highlighting a need for management actions to reduce risks associated with these metals.

Characterizing a rare neurogenetic disease, SLC13A5 citrate transporter disorder, utilizing clinical data in a cloud-based medical record collection system.

Introduction: SLC13A5 citrate transporter disorder is a rare autosomal recessive genetic disease that has a constellation of neurologic symptoms. To better characterize the neurologic and clinical laboratory phenotype, we utilized patient medical records collected by Ciitizen, an Invitae company, with support from the TESS Research Foundation. Methods: Medical records for 15 patients with a suspected genetic and clinical diagnosis of SLC13A5 citrate transporter disorder were collected by Ciitizen, an Invitae company. Genotype, clinical phenotypes, and laboratory data were extracted and analyzed. Results: The 15 patients reported all had epilepsy and global developmental delay. Patients continued to attain motor milestones, though much later than their typically developing peers. Clinical diagnoses support abnormalities in communication, and low or mixed tone with several movement disorders, including, ataxia and dystonia. Serum citrate was elevated in the 3 patients in whom it was measured; other routine laboratory studies assessing renal, liver and blood function had normal values or no consistent abnormalities. Many electroencephalograms (EEGs) were performed (1 to 35 per patient), and most but not all were abnormal, with slowing and/or epileptiform activity. Fourteen of the patients had one or more brain magnetic resonance imaging (MRI) reports: 7 patients had at least one normal brain MRI, but not with any consistent findings except white matter signal changes. Discussion: These results show that in addition to the epilepsy phenotype, SLC13A5 citrate transporter disorder impacts global development, with marked abnormalities in motor abilities, tone, coordination, and communication skills. Further, utilizing cloud-based medical records allows industry, academic, and patient advocacy group collaboration to provide preliminary characterization of a rare genetic disorder. Additional characterization of the neurologic phenotype will be critical to future study and developing treatment for this and related rare genetic disorders.

Pediatric Chemotherapy Infusions in Outpatient Examination Rooms: A Novel Patient Care Approach.

Background: Many health care organizations offer pediatric infusions in outpatient infusion centers or, as in our organization, in a hospital-based outpatient Pediatric Infusion Therapy Center (PITC). When restrictions related to the COVID-19 pandemic decreased our PITC appointment capacity by 40%, other patient and family satisfaction issues were exacerbated. We implemented a new approach to pediatric infusions with the aim of improving patient and family satisfaction and reducing the amount of time in an appointment itinerary without negatively affecting patient safety. Methods: Our team used a phased approach to pilot the administration of short chemotherapy infusions in the same outpatient clinic examination rooms where consultation and routine office visits were conducted. Patients saw their specialist for an examination and, if clinically indicated, their infusion was administered in the same room. Appointment itineraries were then completed. The team tracked efficiency, satisfaction, and safety metrics related to the new process. Results: All efficiency metrics improved. No harm came to the 49 unique patients who received a total of 184 infusions. Patient appointment itineraries were shortened by an average of 1.03 hr. Satisfaction survey responses indicated a clear preference (93%) for the new process. Discussion: The novel approach of offering short infusions in outpatient clinic examination rooms provides an opportunity to ease capacity constraints and further increase patient and family satisfaction. This method may be especially helpful for health care organizations when external influences (e.g., lack of physical space, challenging patient volumes, and pandemics) necessitate a change.

Dermal appendage-dependent patterning of zebrafish atoh1a+ Merkel cells.

Touch system function requires precise interactions between specialized skin cells and somatosensory axons, as exemplified by the vertebrate mechanosensory Merkel cell-neurite complex. Development and patterning of Merkel cells and associated neurites during skin organogenesis remain poorly understood, partly due to the in utero development of mammalian embryos. Here, we discover Merkel cells in the zebrafish epidermis and identify Atonal homolog 1a (Atoh1a) as a marker of zebrafish Merkel cells. We show that zebrafish Merkel cells derive from basal keratinocytes, express neurosecretory and mechanosensory machinery, extend actin-rich microvilli, and complex with somatosensory axons, all hallmarks of mammalian Merkel cells. Merkel cells populate all major adult skin compartments, with region-specific densities and distribution patterns. In vivo photoconversion reveals that Merkel cells undergo steady loss and replenishment during skin homeostasis. Merkel cells develop concomitant with dermal appendages along the trunk and loss of Ectodysplasin signaling, which prevents dermal appendage formation, reduces Merkel cell density by affecting cell differentiation. By contrast, altering dermal appendage morphology changes the distribution, but not density, of Merkel cells. Overall, our studies provide insights into touch system maturation during skin organogenesis and establish zebrafish as an experimentally accessible in vivo model for the study of Merkel cell biology.

Acute Toxicity of 6PPD-Quinone to Early Life Stage Juvenile Chinook (Oncorhynchus tshawytscha) and Coho (Oncorhynchus kisutch) Salmon.

The breakdown product of the rubber tire antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD)-6-PPD-quinone has been strongly implicated in toxic injury and death in coho salmon (Oncorhynchus kisutch) in urban waterways. Whereas recent studies have reported a wide range of sensitivity to 6PPD-quinone in several fish species, little is known about the risks to Chinook salmon (Oncorhynchus tshawytscha), the primary prey of endangered Southern Resident killer whales (Orcinus orca) and the subject of much concern. Chinook face numerous conservation threats in Canada and the United States, with many populations assessed as either endangered or threatened. We evaluated the acute toxicity of 6PPD-quinone to newly feeding (~3 weeks post swim-up) juvenile Chinook and coho. Juvenile Chinook and coho were exposed for 24 h under static conditions to five concentrations of 6PPD-quinone. Juvenile coho were 3 orders of magnitude more sensitive to 6PPD-quinone compared with juvenile Chinook, with 24-h median lethal concentration (LC50) estimates of 41.0 and more than 67 307 ng/L, respectively. The coho LC50 was 2.3-fold lower than what was previously reported for 1+-year-old coho (95 ng/L), highlighting the value of evaluating age-related differences in sensitivity to this toxic tire-related chemical. Both fish species exhibited typical 6PPD-quinone symptomology (gasping, increased ventilation, loss of equilibrium, erratic swimming), with fish that were symptomatic generally exhibiting mortality. The LC50 values derived from our study for coho are below concentrations that have been measured in salmon-bearing waterways, suggesting the potential for population-level consequences in urban waters. The higher relative LC50 values for Chinook compared with coho merits further investigation, including for the potential for population-relevant sublethal effects. Environ Toxicol Chem 2023;42:815-822. © 2023 His Majesty the King in Right of Canada and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Fisheries and Oceans Canada.

Declining concentrations of chlorinated paraffins in endangered St. Lawrence Estuary belugas (Delphinapterus leucas): Response to regulations or a change in diet?

Very high levels of industrial contaminants in St. Lawrence Estuary (SLE) beluga whales represent one of the major threats to this population classified as endangered under the Species at Risk Act in Canada. Elevated concentrations of short-chained chlorinated paraffins (SCCPs) were recently reported in blubber of adult male SLE belugas. Recent regulations for SCCPs in North America, combined with their replacement by medium- (MCCPs) and long-chained chlorinated paraffins (LCCPs), highlight the importance of tracking this toxic chemical class. The objectives of this study were to evaluate (1) levels and profiles of chlorinated paraffins (CPs) in samples obtained from carcasses of adult male, adult female, juvenile, newborn, and fetus beluga, and (2) trends in adult male belugas between 1997 and 2018. Factors potentially influencing CP temporal trends such as age, feeding ecology and sampling year were also explored. SCCPs dominated (64 to 100%) total CP concentrations across all age and sex classes, MCCPs accounted for the remaining proportion of total CPs, and LCCPs were not detected in any sample. The chlorinated paraffin homolog that dominated the most in beluga blubber was C12Cl8. Adult male SCCP concentrations from this study were considerably lower (> 2000-fold) than those recently reported in Simond et al. (2020), likely reflecting a previously erroneous overestimate due to the lack of a suitable analytical method for SCCPs at the time. Both SCCPs and total CPs declined over time in adult males in our study (rate of 1.67 and 1.33% per year, respectively), presumably due in part to the implementation of regulations in 2012. However, there is a need to better understand the possible contribution of a changing diet to contaminant exposure, as stable isotopic ratios of carbon also changed over time.

mTORC2 Loss in Oligodendrocyte Progenitor Cells Results in Regional Hypomyelination in the Central Nervous System.

In the CNS, oligodendrocyte progenitor cells (OPCs) differentiate into mature oligodendrocytes to generate myelin, an essential component for normal nervous system function. OPC differentiation is driven by signaling pathways, such as mTOR, which functions in two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), containing Raptor or Rictor, respectively. In the current studies, mTORC2 signaling was selectively deleted from OPCs in PDGFRα-Cre X Rictorfl/fl mice. This study examined developmental myelination in male and female mice, comparing the impact of mTORC2 deletion in the corpus callosum and spinal cord. In both regions, Rictor loss in OPCs resulted in early reduction in myelin RNAs and proteins. However, these deficits rapidly recovered in spinal cord, where normal myelin was noted at P21 and P45. By contrast, the losses in corpus callosum resulted in severe hypomyelination and increased unmyelinated axons. The hypomyelination may result from decreased oligodendrocytes in the corpus callosum, which persisted in animals as old as postnatal day 350. The current studies focus on uniquely altered signaling pathways following mTORC2 loss in developing oligodendrocytes. A major mTORC2 substrate is phospho-Akt-S473, which was significantly reduced throughout development in both corpus callosum and spinal cord at all ages measured, yet this had little impact in spinal cord. Loss of mTORC2 signaling resulted in decreased expression of actin regulators, such as gelsolin in corpus callosum, but only minimal loss in spinal cord. The current study establishes a regionally specific role for mTORC2 signaling in OPCs, particularly in the corpus callosum.SIGNIFICANCE STATEMENT mTORC1 and mTORC2 signaling has differential impact on myelination in the CNS. Numerous studies identify a role for mTORC1, but deletion of Rictor (mTORC2 signaling) in late-stage oligodendrocytes had little impact on myelination in the CNS. However, the current studies establish that deletion of mTORC2 signaling from oligodendrocyte progenitor cells results in reduced myelination of brain axons. These studies also establish a regional impact of mTORC2, with little change in spinal cord in these conditional Rictor deletion mice. Importantly, in both brain and spinal cord, mTORC2 downstream signaling targets were impacted by Rictor deletion. Yet, these signaling changes had little impact on myelination in spinal cord, while they resulted in long-term alterations in myelination in brain.

Automated, High-Throughput Analysis of Tire-Derived p-Phenylenediamine Quinones (PPDQs) in Water by Online Membrane Sampling Coupled to MS/MS.

The tire-derived contaminant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ) was recently identified as a potent toxin to coho salmon (Oncorhynchus kisutch). Studies investigating 6-PPDQ have employed solid-phase extraction (SPE) or liquid-liquid extraction (LLE) with liquid chromatography-mass spectrometry (LC-MS), providing excellent sensitivity and selectivity. However, cleanup and pre-enrichment steps (SPE/LLE) followed by chromatographic separation can be time- and cost-intensive, limiting sample throughput. The ubiquitous distribution of 6-PPDQ necessitates numerous measurements to identify hotspots for targeted mitigation. We recently developed condensed phase membrane introduction mass spectrometry (CP-MIMS) for rapid 6-PPDQ analysis (2.5 min/sample), with a simple workflow and low limit of detection (8 ng/L). Here, we describe improved quantitation using isotopically labeled internal standards and inclusion of a suite of PPDQ analogues. A low-cost autosampler and data processing software were developed from a three-dimensional (3D) printer and Matlab to fully realize the high-throughput capabilities of CP-MIMS. Cross-validation with a commercial LC-MS method for 10 surface waters provides excellent agreement (slope: 1.01; R2 = 0.992). We employ this analytical approach to probe fundamental questions regarding sample stability and sorption of 6-PPDQ under lab-controlled conditions. Further, the results for 192 surface water samples provide the first spatiotemporal characterization of PPDQs on Vancouver Island and the lower mainland of British Columbia.

A Multi-Matrix Metabolomic Approach in Ringed Seals and Beluga Whales to Evaluate Contaminant and Climate-Related Stressors.

As a high trophic-level species, ringed seals (Pusa hispida) and beluga whales (Delphinapterus leucas) are particularly vulnerable to elevated concentrations of biomagnifying contaminants, such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and mercury (Hg). These species also face climate-change-related impacts which are leading to alterations in their diet and associated contaminant exposure. The metabolomic profile of marine mammal tissues and how it changes to environmental stressors is poorly understood. This study characterizes the profiles of 235 metabolites across plasma, liver, and inner and outer blubber in adult ringed seals and beluga whales and assesses how these profiles change as a consequence of contaminants and dietary changes. In both species, inner and outer blubber were characterized by a greater proportion of lipid classes, whereas the dominant metabolites in liver and plasma were amino acids, carbohydrates, biogenic amines and lysophosphatidylcholines. Several metabolite profiles in ringed seal plasma correlated with δ13C, while metabolite profiles in blubber were affected by hexabromobenzene in ringed seals and PBDEs and Hg in belugas. This study provides insight into inter-matrix similarities and differences across tissues and suggests that plasma and liver are more suitable for studying changes in diet, whereas liver and blubber are more suitable for studying the impacts of contaminants.

Characterization and Interpolation of Sediment Polychlorinated Biphenyls and Polybrominated Diphenyl Ethers in Resident Killer Whale Habitat along the Coast of British Columbia, Canada.

The northeastern Pacific northern and southern resident killer whale (Orcinus orca) populations are listed as threatened and endangered in Canada, respectively, with persistent, bioaccumulative contaminants, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs), posing threats to their recovery. Concentrations of PCBs and PBDEs in subtidal surface sediments collected from 97 sites along the British Columbia (BC) coast were used to identify their distribution and profiles, and to assess killer whale habitat quality. Victoria Harbour (VH3(site ID: 1) ) sediments exhibited the highest PCB and PBDE concentrations. For PCBs, PCB-138 was found at the highest concentration, followed by PCB-153, PCB-110, PCB-149, PCB-101, and PCB-118. For PBDEs, individual congeners were ranked as follows: BDE-209 > BDE-207 > BDE-206 > BDE-208 > BDE-47 > BDE-99. Principal component analyses (PCA) illustrated the variations in contaminant profiles, with PC1 for PCBs and PBDEs correlated with the octanol-water partition coefficient (log KOW , p < 0.003). Based on the PCA, sediment particle size, total organic carbon (TOC), and water depth at collection were other factors associated with the distribution of PBDEs, while PCB profiles were associated with TOC. Total PCB and PBDE concentrations at 100% and 34% of the sites, respectively, exceeded the recently adopted British Columbia's Ministry of Environment and Climate Change Strategy Working Sediment Quality Guidelines (PCBs 3.7 pg/g dry wt and PBDEs 1000 pg/g dry wt), considered protective of killer whales. Our findings suggest that the legacy of banned PCBs and PBDEs has the potential to constrain the recovery of killer whales as a result of their mobilization from sediments and consequent uptake by marine food webs. Environ Toxicol Chem 2022;41:2139-2151. © 2022 SETAC.

Contributions and perspectives of Indigenous Peoples to the study of mercury in the Arctic.

Arctic Indigenous Peoples are among the most exposed humans when it comes to foodborne mercury (Hg). In response, Hg monitoring and research have been on-going in the circumpolar Arctic since about 1991; this work has been mainly possible through the involvement of Arctic Indigenous Peoples. The present overview was initially conducted in the context of a broader assessment of Hg research organized by the Arctic Monitoring and Assessment Programme. This article provides examples of Indigenous Peoples' contributions to Hg monitoring and research in the Arctic, and discusses approaches that could be used, and improved upon, when carrying out future activities. Over 40 mercury projects conducted with/by Indigenous Peoples are identified for different circumpolar regions including the U.S., Canada, Greenland, Sweden, Finland, and Russia as well as instances where Indigenous Knowledge contributed to the understanding of Hg contamination in the Arctic. Perspectives and visions of future Hg research as well as recommendations are presented. The establishment of collaborative processes and partnership/co-production approaches with scientists and Indigenous Peoples, using good communication practices and transparency in research activities, are key to the success of research and monitoring activities in the Arctic. Sustainable funding for community-driven monitoring and research programs in Arctic countries would be beneficial and assist in developing more research/monitoring capacity and would promote a more holistic approach to understanding Hg in the Arctic. These activities should be well connected to circumpolar/international initiatives to ensure broader availability of the information and uptake in policy development.

p70S6 kinase regulates oligodendrocyte differentiation and is active in remyelinating lesions.

The p70 ribosomal S6 kinases (p70 ribosomal S6 kinase 1 and p70 ribosomal S6 kinase 2) are downstream targets of the mechanistic target of rapamycin signalling pathway. p70 ribosomal S6 kinase 1 specifically has demonstrated functions in regulating cell size in Drosophila and in insulin-sensitive cell populations in mammals. Prior studies demonstrated that the mechanistic target of the rapamycin pathway promotes oligodendrocyte differentiation and developmental myelination; however, how the immediate downstream targets of mechanistic target of rapamycin regulate these processes has not been elucidated. Here, we tested the hypothesis that p70 ribosomal S6 kinase 1 regulates oligodendrocyte differentiation during developmental myelination and remyelination processes in the CNS. We demonstrate that p70 ribosomal S6 kinase activity peaks in oligodendrocyte lineage cells at the time when they transition to myelinating oligodendrocytes during developmental myelination in the mouse spinal cord. We further show p70 ribosomal S6 kinase activity in differentiating oligodendrocytes in acute demyelinating lesions induced by lysophosphatidylcholine injection or by experimental autoimmune encephalomyelitis in mice. In demyelinated lesions, the expression of the p70 ribosomal S6 kinase target, phosphorylated S6 ribosomal protein, was transient and highest in maturing oligodendrocytes. Interestingly, we also identified p70 ribosomal S6 kinase activity in oligodendrocyte lineage cells in active multiple sclerosis lesions. Consistent with its predicted function in promoting oligodendrocyte differentiation, we demonstrate that specifically inhibiting p70 ribosomal S6 kinase 1 in cultured oligodendrocyte precursor cells significantly impairs cell lineage progression and expression of myelin basic protein. Finally, we used zebrafish to show in vivo that inhibiting p70 ribosomal S6 kinase 1 function in oligodendroglial cells reduces their differentiation and the number of myelin internodes produced. These data reveal an essential function of p70 ribosomal S6 kinase 1 in promoting oligodendrocyte differentiation during development and remyelination across multiple species.

Growth and Overall Health of Patients with SLC13A5 Citrate Transporter Disorder.

We were interested in elucidating the non-neurologic health of patients with autosomal recessive SLC13A5 Citrate Transporter (NaCT) Disorder. Multiple variants have been reported that cause a loss of transporter activity, resulting in significant neurologic impairment, including seizures, as well as motor and cognitive dysfunction. Additionally, most patients lack tooth enamel (amelogenesis imperfecta). However, patients have not had their overall health and growth described in detail. Here we characterized the non-neurologic health of 15 patients with medical records uploaded to Ciitizen, a cloud-based patient medical records portal. Ciitizen used a query method for data extraction. Overall, the patients' records suggested a moderate number of gastrointestinal issues related to feeding, reflux, vomiting and weight gain and a diverse number of respiratory complaints. Other organ systems had single or no abnormal diagnoses, including liver, renal and cardiac. Growth parameters were mostly in the normal range during early life, with a trend toward slower growth in the few adolescent patients with data available. The gastrointestinal and pulmonary issues may at least partially be explained by the severity of the neurologic disorder. More data are needed to clarify if growth is impacted during adolescence and if adult patients develop or are protected from non-neurologic disorders.

Neuropsychological Functioning in Alexander Disease: A Case Series.

Limited information is known about neuropsychological outcomes in Alexander disease, a rare leukodystrophy. Two pediatric cases are summarized. Case 1 (evaluations at 6, 7, 9, and 12 years of age) represents Type I Alexander disease with associated seizures. Case 2 (evaluations at 12, 13, and 16 years of age) represents Type II Alexander disease without additional complications. Case 1 experienced declines in intellectual functioning, visual motor skills, receptive vocabulary, verbal memory, and academic achievement. Case 2 experienced variable neurocognitive change and academic functioning, with average word reading and spelling. Verbal memory also remained intact. Taken together, individuals with Alexander disease may experience cognitive decline to variable degrees. Type I Alexander disease, associated with earlier onset and additional neurological complications, may presage greater cognitive decline than Type II. Due to variability in functioning over time, it is critical to follow individuals across development to make recommendations for educational and treatment planning.

Characterization of the microbiome and immune response in corals with chronic Montipora white syndrome.

Coral diseases have increased in frequency and intensity around the tropics worldwide. However, in many cases, little is known about their etiology. Montipora white syndrome (MWS) is a common disease affecting the coral Montipora capitata, a major reef builder in Hawai'i. Chronic Montipora white syndrome (cMWS) is a slow-moving form of the disease that affects M. capitata throughout the year. The effects of this chronic disease on coral immunology and microbiology are currently unknown. In this study, we use prophenoloxidase immune assays and 16S rRNA gene amplicon sequencing to characterize the microbiome and immunological response associated with cMWS. Our results show that immunological and microbiological responses are highly localized. Relative to diseased samples, apparently healthy portions of cMWS corals differed in immune activity and in the relative abundance of microbial taxa. Coral tissues with cMWS showed decreased tyrosinase-type catecholase and tyrosinase-type cresolase activity and increased laccase-type activity. Catecholase and cresolase activity were negatively correlated across all tissue types with microbiome richness. The localized effect of cMWS on coral microbiology and immunology is probably an important reason for the slow progression of the disease. This local confinement may facilitate interventions that focus on localized treatments on tissue types. This study provides an important baseline to understand the interplay between the microbiome and immune system and the mechanisms used by corals to manage chronic microbial perturbations associated with white syndrome.

PAK1 Positively Regulates Oligodendrocyte Morphology and Myelination.

The actin cytoskeleton is crucial for oligodendrocyte differentiation and myelination. Here we show that p21-activated kinase 1 (PAK1), a well-known actin regulator, promotes oligodendrocyte morphologic change and myelin production in the CNS. A combination of in vitro and in vivo models demonstrated that PAK1 is expressed throughout the oligodendrocyte lineage with highest expression in differentiated oligodendrocytes. Inhibiting PAK1 early in oligodendrocyte development decreased oligodendrocyte morphologic complexity and altered F-actin spreading at the tips of oligodendrocyte progenitor cell processes. Constitutively activating AKT in oligodendrocytes in male and female mice, which leads to excessive myelin wrapping, increased PAK1 expression, suggesting an impact of PAK1 during active myelin wrapping. Furthermore, constitutively activating PAK1 in oligodendrocytes in zebrafish led to an increase in myelin internode length while inhibiting PAK1 during active myelination decreased internode length. As myelin parameters influence conduction velocity, these data suggest that PAK1 may influence communication within the CNS. These data support a model in which PAK1 is a positive regulator of CNS myelination.SIGNIFICANCE STATEMENT Myelin is a critical component of the CNS that provides metabolic support to neurons and also facilitates communication between cells in the CNS. Recent data demonstrate that actin dynamics drives myelin wrapping, but how actin is regulated during myelin wrapping is unknown. The authors investigate the role of the cytoskeletal modulator PAK1 during differentiation and myelination by oligodendrocytes, the myelinating cells of the CNS. They demonstrate that PAK1 promotes oligodendrocyte differentiation and myelination by modulating the cytoskeleton and thereby internode length, thus playing a critical role in the function of the CNS.

The contribution of qualitative research within the PRECISE study in sub-Saharan Africa.

The PRECISE Network is a cohort study established to investigate hypertension, fetal growth restriction and stillbirth (described as "placental disorders") in Kenya, Mozambique and The Gambia. Several pregnancy or birth cohorts have been set up in low- and middle-income countries, focussed on maternal and child health. Qualitative research methods are sometimes used alongside quantitative data collection from these cohorts. Researchers affiliated with PRECISE are also planning to use qualitative methods, from the perspective of multiple subject areas. This paper provides an overview of the different ways in which qualitative research methods can contribute to achieving PRECISE's objectives, and discusses the combination of qualitative methods with quantitative cohort studies more generally.We present planned qualitative work in six subject areas (health systems, health geography, mental health, community engagement, the implementation of the TraCer tool, and respectful maternity care). Based on these plans, with reference to other cohort studies on maternal and child health, and in the context of the methodological literature on mixed methods approaches, we find that qualitative work may have several different functions in relation to cohort studies, including informing the quantitative data collection or interpretation. Researchers may also conduct qualitative work in pursuit of a complementary research agenda. The degree to which integration between qualitative and quantitative methods will be sought and achieved within PRECISE remains to be seen. Overall, we conclude that the synergies resulting from the combination of cohort studies with qualitative research are an asset to the field of maternal and child health.

Mechanistic Target of Rapamycin Regulates the Oligodendrocyte Cytoskeleton during Myelination.

During differentiation, oligodendrocyte precursor cells (OPCs) extend a network of processes that make contact with axons and initiate myelination. Recent studies revealed that actin polymerization is required for initiation of myelination whereas actin depolymerization promotes myelin wrapping. Here, we used primary OPCs in culture isolated from neonatal rat cortices of both sexes and young male and female mice with oligodendrocyte-specific deletion of mechanistic target of rapamycin (mTOR) to demonstrate that mTOR regulates expression of specific cytoskeletal targets and actin reorganization in oligodendrocytes during developmental myelination. Loss or inhibition of mTOR reduced expression of profilin2 and ARPC3, actin polymerizing factors, and elevated levels of active cofilin, which mediates actin depolymerization. The deficits in actin polymerization were revealed in reduced phalloidin and deficits in oligodendrocyte cellular branching complexity at the peak of morphologic differentiation and a delay in initiation of myelination. We further show a critical role for mTOR in expression and localization of myelin basic protein (Mbp) mRNA and MBP protein to the cellular processes where it is necessary at the myelin membrane for axon wrapping. Mbp mRNA transport deficits were confirmed by single molecule RNA FISH. Moreover, expression of the kinesin family member 1B, an Mbp mRNA transport protein, was reduced in CC1+ cells in the mTOR cKO and in mTOR inhibited oligodendrocytes undergoing differentiation in vitro These data support the conclusion that mTOR regulates both initiation of myelination and axon wrapping by targeting cytoskeletal reorganization and MBP localization to oligodendrocyte processes.SIGNIFICANCE STATEMENT Myelination is essential for normal CNS development and adult axon preservation and function. The mechanistic target of rapamycin (mTOR) signaling pathway has been implicated in promoting CNS myelination; however, there is a gap in our understanding of the mechanisms by which mTOR promotes developmental myelination through regulating specific downstream targets. Here, we present evidence that mTOR promotes the initiation of myelination through regulating specific cytoskeletal targets and cellular process expansion by oligodendrocyte precursor cells as well as expression and cellular localization of myelin basic protein.