Transcriptome-wide analyses of early immune responses in lumpfish leukocytes upon stimulation with poly(I:C).

Background: Both bacterial and viral diseases are a major threat to farmed fish. As the antiviral immune mechanisms in lumpfish (Cyclopterus lumpus L.) are poorly understood, lumpfish leukocytes were stimulated with poly(I:C), a synthetic analog of double stranded RNA, which mimic viral infections, and RNA sequencing was performed. Methods: To address this gap, we stimulated lumpfish leukocytes with poly(I:C) for 6 and 24 hours and did RNA sequencing with three parallels per timepoint. Genome guided mapping was performed to define differentially expressed genes (DEGs). Results: Immune genes were identified, and transcriptome-wide analyses of early immune responses showed that 376 and 2372 transcripts were significantly differentially expressed 6 and 24 hours post exposure (hpe) to poly(I:C), respectively. The most enriched GO terms when time had been accounted for, were immune system processes (GO:0002376) and immune response (GO:0006955). Analysis of DEGs showed that among the most highly upregulated genes were TLRs and genes belonging to the RIG-I signaling pathway, including LGP2, STING and MX, as well as IRF3 and IL12A. RIG-I was not identified, but in silico analyses showed that genes encoding proteins involved in pathogen recognition, cell signaling, and cytokines of the TLR and RIG-I signaling pathway are mostly conserved in lumpfish when compared to mammals and other teleost species. Conclusions: Our analyses unravel the innate immune pathways playing a major role in antiviral defense in lumpfish. The information gathered can be used in comparative studies and lay the groundwork for future functional analyses of immune and pathogenicity mechanisms. Such knowledge is also necessary for the development of immunoprophylactic measures for lumpfish, which is extensively cultivated for use as cleaner fish in the aquaculture for removal of sea lice from Atlantic salmon (Salmo salar L.).

Systemic interferon type I and B cell responses are impaired in autoimmune polyendocrine syndrome type 1.

Autoimmune polyendocrine syndrome type I (APS-1) is caused by mutations in the autoimmune regulator (AIRE) gene and characterised clinically by multiple autoimmune manifestations and serologically by autoantibodies against tissue proteins and cytokines. We here hypothesised that lack of AIRE expression in thymus affects blood immune cells and performed whole-blood microarray analysis (N = 16 APS-I patients vs 16 controls), qPCR verification, and bioinformatic deconvolution of cell subsets. We identified B cell responses as being downregulated in APS-1 patients, which was confirmed by qPCR; these results call for further studies on B cells in this disorder. The type I interferon (IFN-I) pathway was also downregulated in APS-1, and the presence of IFN antibodies is the likely reason for this mild overall downregulation of the IFN-I genes in most APS-1 patients.

MERISTEM-DEFECTIVE regulates the balance between stemness and differentiation in the root meristem through RNA splicing control.

Plants respond to environmental stresses through controlled stem cell maintenance and meristem activity. One level of gene regulation is RNA alternative splicing. However, the mechanistic link between stress, meristem function and RNA splicing is poorly understood. The MERISTEM-DEFECTIVE (MDF) Arabidopsis gene encodes an SR-related family protein, required for meristem function and leaf vascularization, and is the likely orthologue of the human SART1 and yeast Snu66 splicing factors. MDF is required for the correct splicing and expression of key transcripts associated with root meristem function. We identified RSZ33 and ACC1, both known to regulate cell patterning, as splicing targets required for MDF function in the meristem. MDF expression is modulated by osmotic and cold stress, associated with differential splicing and specific isoform accumulation and shuttling between nucleus and cytosol, and acts in part via a splicing target SR34. We propose a model in which MDF controls splicing in the root meristem to promote stemness and to repress stress response, cell differentiation and cell death pathways.

USH2A Gene Mutations in Rabbits Lead to Progressive Retinal Degeneration and Hearing Loss.

Purpose: Mutations in USH2A gene are responsible for the greatest proportion of the Usher Syndrome (USH) population, among which more than 30% are frameshift mutations on exon 13. A clinically relevant animal model has been absent for USH2A-related vision loss. Here we sought to establish a rabbit model carrying USH2A frameshift mutation on exon 12 (human exon 13 equivalent). Methods: CRISPR/Cas9 reagents targeting the rabbit USH2A exon 12 were delivered into rabbit embryos to produce an USH2A mutant rabbit line. The USH2A knockout animals were subjected to a series of functional and morphological analyses, including acoustic auditory brainstem responses, electroretinography, optical coherence tomography, fundus photography, fundus autofluorescence, histology, and immunohistochemistry. Results: The USH2A mutant rabbits exhibit hyper-autofluorescent signals on fundus autofluorescence and hyper-reflective signals on optical coherence tomography images as early as 4 months of age, which indicate retinal pigment epithelium damage. Auditory brainstem response measurement in these rabbits showed moderate to severe hearing loss. Electroretinography signals of both rod and cone function were decreased in the USH2A mutant rabbits starting from 7 months of age and further decreased at 15 to 22 months of age, indicating progressive photoreceptor degeneration, which is confirmed by histopathological examination. Conclusions: Disruption of USH2A gene in rabbits is sufficient to induce hearing loss and progressive photoreceptor degeneration, mimicking the USH2A clinical disease. Translational Relevance: To our knowledge, this study presents the first mammalian model of USH2 showing the phenotype of retinitis pigmentosa. This study supports the use of rabbits as a clinically relevant large animal model to understand the pathogenesis and to develop novel therapeutics for Usher syndrome.

Photoreception and transcriptomic response to light during early development of a teleost with a life cycle tightly controlled by seasonal changes in photoperiod.

Light cues vary along the axis of periodicity, intensity and spectrum and perception of light is dependent on the photoreceptive capacity encoded within the genome and the opsins expressed. A global approach was taken to analyze the photoreceptive capacity and the effect of differing light conditions on a developing teleost prior to first feeding. The transcriptomes of embryos and alevins of Atlantic salmon (Salmo salar) exposed to different light conditions were analyzed, including a developmental series and a circadian profile. The results showed that genes mediating nonvisual photoreception are present prior to hatching when the retina is poorly differentiated. The clock genes were expressed early, but the circadian profile showed that only two clock genes were significantly cycling before first feeding. Few genes were differentially expressed between day and night within a light condition; however, many genes were significantly different between light conditions, indicating that light environment has an impact on the transcriptome during early development. Comparing the transcriptome data from constant conditions to periodicity of white light or different colors revealed overrepresentation of genes related to photoreception, eye development, muscle contraction, degradation of metabolites and cell cycle among others, and in constant light, several clock genes were upregulated. In constant white light and periodicity of green light, genes associated with DNA replication, chromatin remodeling, cell division and DNA repair were downregulated. The study implies a direct influence of light conditions on the transcriptome profile at early developmental stages, by a complex photoreceptive system where few clock genes are cycling.

Loss of the chromatin remodeler CHD7 impacts glial cells and myelination in the mouse cochlear spiral ganglion.

CHARGE syndrome is a multiple anomaly developmental disorder characterized by a variety of sensory deficits, including sensorineural hearing loss of unknown etiology. Most cases of CHARGE are caused by heterozygous pathogenic variants in CHD7, the gene encoding Chromodomain DNA-binding Protein 7 (CHD7), a chromatin remodeler important for the development of neurons and glial cells. Previous studies in the Chd7Gt/+ mouse model of CHARGE syndrome showed substantial neuron loss in the early stages of the developing inner ear that are compensated for by mid-gestation. In this study, we sought to determine if early developmental delays caused by Chd7 haploinsufficiency affect neurons, glial cells, and inner hair cell innervation in the mature cochlea. Analysis of auditory brainstem response recordings in Chd7Gt/+ adult animals showed elevated thresholds at 4 kHz and 16 kHz, but no differences in ABR Wave I peak latency or amplitude compared to wild type controls. Proportions of neurons in the Chd7Gt/+ adult spiral ganglion and densities of nerve projections from the spiral ganglion to the organ of Corti were not significantly different from wild type controls. Inner hair cell synapse formation also appeared unaffected in mature Chd7Gt/+ cochleae. However, histological analysis of adult Chd7Gt/+ cochleae revealed diminished satellite glial cells and hypermyelinated Type I spiral ganglion axons. We characterized the expression of CHD7 in developing inner ear glia and found CHD7 to be expressed during a tight window of inner ear development at the Schwann cell precursor stage at E9.5. While cochlear neurons appear to differentiate normally in the setting of Chd7 haploinsufficiency, our results suggest an important role for CHD7 in glial cells in the inner ear. This study highlights the dynamic nature of CHD7 activity during inner ear development in mice and contributes to understanding CHARGE syndrome pathology.

Noise overstimulation of young adult UMHET4 mice accelerates age-related hearing loss.

Many factors contribute to hearing loss commonly found in older adults. There can be natural aging of cellular elements, hearing loss previously induced by environmental factors such as noise or ototoxic drugs as well as genetic and epigenetic influences. Even when noise overstimulation does not immediately cause permanent hearing loss it has recently been shown to increase later age-related hearing loss (ARHL). The present study further investigated this condition in the UMHET4 mouse model by comparing a small arms fire (SAF)-like impulse noise exposure that has the greatest immediate effect in more apical cochlear regions to a broadband noise (BBN) exposure that has the greatest immediate effect in more basal cochlear regions. Both noise exposures were given at levels that only induced temporary auditory brainstem response (ABR) threshold shifts (TS). Mice were noise exposed at 5 months of age followed by ABR assessment at 6, 12, 18, 21, and 24 months of age. Mice that received the SAF-like impulse noise had accelerated age-related TS at 4 kHz that appeared at 12 months of age (significantly increased compared to no-noise controls). This increased TS at 4 kHz continued at 18 and 21 months but was no longer significantly greater at 24 months of age. The SAF-like impulse noise also induced a significantly greater mean TS at 48 kHz, first appearing at 18 months of age and continuing to be significantly greater than controls at 21 and 24 months. The BBN induced a different pace and pattern of enhanced age-related ABR TS. The mean TS for the BBN group first became significantly greater than controls at 18 months of age and only at 48 kHz. It remained significantly greater than controls at 21 months but was no longer significantly greater at 24 months of age. Results, therefore, show different influences on ARHL for the two different noise exposure conditions. Noise-induced enhancement appears to provide more an acceleration than overall total increase in ARHL.

Observation and Mitigation of Lamellar Silica Particles Formed in Pharmaceutical Products Packaged in Glass Vials.

A new type of lamellae-like particles was observed in protein based liquid therapeutic protein drug product (DP) packaged in standard (STD) and delamination controlled (DC) Type IB glass vials stored at 2-8°C as early as two weeks after manufacture. These particles were determined to be remarkably different from lamellae in not only in their chemical composition, but in the mechanism by which these are formed. The lamellae-like particles were an ultra-thin (< 200 nm) film, appeared curled, sheet-like, folded with no defined edges identified as lamellar silica composed of silica and polysorbate 80 (PS 80). It was also observed that the lamellar silica particles, when formed in a given drug product lot, not only were observed in a small percentage of vials, but also remained at low (≤ 5) numbers in affected vials, often decreasing in number over time. This is in contrast to the large number of commonly reported glass lamellae (hundreds per vial) observed in vials prone to delamination with a glass vial interior showing a delaminated inner surface. In this case study, evidence from low Si leachable levels in solution and various surface analytical techniques supported the conclusion that there was neither delamination nor early signs of glass delamination like reaction zones occurring in those impacted vials, regardless. A mechanism for particle formation was hypothesized and experimentally confirmed. Lamellar silica particles are composed of an admixture of condensed silica and PS 80 deposited on the interior walls of glass vials, which form and may be released into solution over time. The root cause was determined to be conditions present during preparation of the vials for drug product filling, specifically the vial washing and depyrogenation steps. These conditions are known to make glass vials prone to delamination; in this case study, they resulted in interactions between the glass and PS 80 present in the formulation. Incomplete drying of the glass vials during depyrogenation in closed ovens was confirmed as the contributing factors that led to lamellar silica particle formation via the studies of silicate spiked into the DC Type IB glass vials filled with the mAb DP in which lamellar silica particles were observed. Prevention of lamellar silica particles formation was successfully achieved through optimization of the duration and pressure of air blow during the vial washing and drying process in a depyrogenation oven. This was evidenced by the lack of appearance of the lamellar silica particles over 48 months for the DP lots filled post optimization. Additionally, the formation of lamellar silica was also mitigated by changing the vial washing process from a closed oven process to a tunnel process, which allowed for improved air flow and hence better drying of the vial primary container.

Setting impurity limits for endogenous substances: Recommendations for a harmonized procedure and an example using fatty acids.

Endogenous substances, such as fatty, amino, and nucleic acids, are often purposefully used in parenterally pharmaceuticals, but may be present as impurities. Currently, no consensus guidance exists on setting impurity limits for these substances. Specific procedures are needed, as the amount and types of toxicity data available for endogenous substances are typically far less than those for other chemical impurities. Additionally, the parenteral route of administration of these substances is inherently non-physiological, resulting in potentially different or increased severity of toxicity. Risk Assessment Process Maps (RAPMAPs) are proposed as a model to facilitate the development of health-based exposure limits (HBELs) for endogenous substances. This yielded a framework that was applied to derive HBELs for several fatty acids commonly used in parenteral pharmaceuticals. This approach was used to derive HBELs with further vetting based on anticipated perturbations in physiological serum levels, impacts of dose-rate, and consideration of intermittent dosing. Parenteral HBELs of 100-500 mg/day were generated for several fatty acids, and a proposed class-based limit of 50 mg/day to be used in the absence of chemical-specific data. This default limit is consistent with the low toxicity of this chemical class and ICH Q3C value for Class 3 solvents.

An Evaluation of the Occupational Health Hazards of Peptide Couplers.

Peptide couplers (also known as amide bond-forming reagents or coupling reagents) are broadly used in organic chemical syntheses, especially in the pharmaceutical industry. Yet, occupational health hazards associated with this chemical class are largely unexplored, which is disconcerting given the intrinsic reactivity of these compounds. Several case studies involving occupational exposures reported adverse respiratory and dermal health effects, providing initial evidence of chemical sensitization. To address the paucity of toxicological data, a pharmaceutical cross-industry task force was formed to evaluate and assess the potential of these compounds to cause eye and dermal irritation as well as corrosivity and dermal sensitization. The goal of our work was to inform health and safety professionals as well as pharmaceutical and organic chemists of the occupational health hazards associated with this chemical class. To that end, 25 of the most commonly used peptide couplers and five hydrolysis products were selected for in vivo, in vitro, and in silico testing. Our findings confirmed that dermal sensitization is a concern for this chemical class with 21/25 peptide couplers testing positive for dermal sensitization and 15 of these being strong/extreme sensitizers. We also found that dermal corrosion and irritation (8/25) as well as eye irritation (9/25) were health hazards associated with peptide couplers and their hydrolysis products (4/5 were dermal irritants or corrosive and 4/5 were eye irritants). Resulting outcomes were synthesized to inform decision making in peptide coupler selection and enable data-driven hazard communication to workers. The latter includes harmonized hazard classifications, appropriate handling recommendations, and accurate safety data sheets, which support the industrial hygiene hierarchy of control strategies and risk assessment. Our study demonstrates the merits of an integrated, in vivo -in silico analysis, applied here to the skin sensitization endpoint using the Computer-Aided Discovery and REdesign (CADRE) and Derek Nexus programs. We show that experimental data can improve predictive models by filling existing data gaps while, concurrently, providing computational insights into key initiating events and elucidating the chemical structural features contributing to adverse health effects. This interactive, interdisciplinary approach is consistent with Green Chemistry principles that seek to improve the selection and design of less hazardous reagents in industrial processes and applications.

Effects of Calcitonin-Gene-Related-Peptide on Auditory Nerve Activity.

Calcitonin-gene-related peptide (CGRP) is a lateral olivocochlear (LOC) efferent neurotransmitter. Depression of sound-driven auditory brainstem response amplitude in CGRP-null mice suggests the potential for endogenous CGRP release to upregulate spontaneous and/or sound-driven auditory nerve (AN) activity. We chronically infused CGRP into the guinea pig cochlea and evaluated changes in AN activity as well as outer hair cell (OHC) function. The amplitude of both round window noise (a measure of ensemble spontaneous activity) and the synchronous whole-nerve response to sound (compound action potential, CAP) were enhanced. Lack of change in both onset adaptation and steady state amplitude of sound-evoked distortion product otoacoustic emission (DPOAE) responses indicated CGRP had no effect on OHCs, suggesting the origin of the observed changes was neural. Combined with results from the CGRP-null mice, these results appear to confirm that endogenous CGRP enhances auditory nerve activity when released by the LOC neurons. However, infusion of the CGRP receptor antagonist CGRP (8-37) did not reliably influence spontaneous or sound-driven AN activity, or OHC function, results that contrast with the decreased ABR amplitude measured in CGRP-null mice.

GJB2 gene therapy and conditional deletion reveal developmental stage-dependent effects on inner ear structure and function.

Pathogenic variants in GJB2, the gene encoding connexin 26, are the most common cause of autosomal-recessive hereditary deafness. Despite this high prevalence, pathogenic mechanisms leading to GJB2-related deafness are not well understood, and cures are absent. Humans with GJB2-related deafness retain at least some auditory hair cells and neurons, and their deafness is usually stable. In contrast, mice with conditional loss of Gjb2 in supporting cells exhibit extensive loss of hair cells and neurons and rapidly progress to profound deafness, precluding the application of therapies that require intact cochlear cells. In an attempt to design a less severe Gjb2 animal model, we generated mice with inducible Sox10iCre ERT2 -mediated loss of Gjb2. Tamoxifen injection led to reduced connexin 26 expression and impaired function, but cochlear hair cells and neurons survived for 2 months, allowing phenotypic rescue attempts within this time. AAV-mediated gene transfer of GJB2 in mature mutant ears did not demonstrate threshold improvement and in some animals exacerbated hearing loss and resulted in hair cell loss. We conclude that Sox10iCre ERT2 ;Gjb2 flox/flox mice are valuable for studying the biology of connexin 26 in the cochlea. In particular, these mice may be useful for evaluating gene therapy vectors and development of therapies for GJB2-related deafness.

Rapamycin Added to Diet in Late Mid-Life Delays Age-Related Hearing Loss in UMHET4 Mice.

Our previous study demonstrated rapamycin added to diet at 4 months of age had significantly less age-related outer hair cell loss in the basal half of the cochlea at 22 months of age compared to mice without rapamycin. The present study tested adding rapamycin to diet later in life, at 14 months of age, and added a longitudinal assessment of auditory brain stem response (ABR). The present study used UMHET4 mice, a 4 way cross in which all grandparental strains lack the Cdh23753A allele that predisposes to early onset, progressive hearing loss. UMHET4 mice typically have normal hearing until 16-17 months, then exhibit threshold shifts at low frequencies/apical cochlea and later in more basal high frequency regions. ABR thresholds at 4, 12, 24, and 48 kHz were assessed at 12, 18, and 24 months of age and compared to baseline ABR thresholds acquired at 5 months of age to determine threshold shifts (TS). There was no TS at 12 months of age at any frequency tested. At 18 months of age mice with rapamycin added to diet at 14 months had a significantly lower mean TS at 4 and 12 kHz compared to mice on control diet with no significant difference at 24 and 48 kHz. At 24 months of age, the mean 4 kHz TS in rapamycin diet group was no longer significantly lower than the control diet group, while the 12 kHz mean remained significantly lower. Mean TS at 24 and 48 kHz in the rapamycin diet group became significantly lower than in the control diet group at 24 months. Hair cell counts at 24 months showed large loss in the apical half of most rapamycin and control diet mice cochleae with no significant difference between groups. There was only mild outer hair cell loss in the basal half of rapamycin and control diet mice cochleae with no significant difference between groups. The results show that a later life addition of rapamycin can decrease age-related hearing loss in the mouse model, however, it also suggests that this decrease is a delay/deceleration rather than a complete prevention.

Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner.

Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease.

Potential Transcriptional Biomarkers to Guide Glucocorticoid Replacement in Autoimmune Addison's Disease.

BACKGROUND: No reliable biomarkers exist to guide glucocorticoid (GC) replacement treatment in autoimmune Addison's disease (AAD), leading to overtreatment with alarming and persistent side effects or undertreatment, which could be fatal. OBJECTIVE: To explore changes in gene expression following different GC replacement doses as a means of identifying candidate transcriptional biomarkers to guide GC replacement in AAD. METHODS: Step 1: Global microarray expression analysis on RNA from whole blood before and after intravenous infusion of 100 mg hydrocortisone (HC) in 10 patients with AAD. In 3 of the most highly upregulated genes, we performed real-time PCR (rt-PCR) to compare gene expression levels before and 3, 4, and 6 hours after the HC infusion. Step 2: Rt-PCR to compare expression levels of 93 GC-regulated genes in normal versus very low morning cortisol levels in 27 patients with AAD. RESULTS: Step 1: Two hours after infusion of 100 mg HC, there was a marked increase in FKBP5, MMP9, and DSIPI expression levels. MMP9 and DSIPI expression levels correlated with serum cortisol. Step 2: Expression levels of CEBPB, DDIT4, FKBP5, DSIPI, and VDR were increased and levels of ADARB1, ARIDB5, and POU2F1 decreased in normal versus very low morning cortisol. Normal serum cortisol levels positively correlated with DSIPI, DDIT4, and FKBP5 expression. CONCLUSIONS: We introduce gene expression as a novel approach to guide GC replacement in AAD. We suggest that gene expression of DSIPI, DDIT4, and FKBP5 are particularly promising candidate biomarkers of GC replacement, followed by MMP9, CEBPB, VDR, ADARB1, ARID5B, and POU2F1.

Considerations when deriving compound-specific limits for extractables and leachables from pharmaceutical products: Four case studies.

Leachables from pharmaceutical container closure systems are a subset of impurities that present in drug products and may pose a risk to patients or compromise product quality. Extractable studies can identify potential leachables, and extractables and leachables (E&Ls) should be evaluated during development of the impurity control strategy. Currently, there is a lack of specific regulatory guidance on how to risk assess E&Ls; this may lead to inconsistency across the industry. This manuscript is a cross-industry Extractables and Leachables Safety Information Exchange (ELSIE) consortium collaboration and follow-up to Broschard et al. (2016), which aims to provide further clarity and detail on the conduct of E&L risk assessments. Where sufficient data are available, a health-based exposure limit termed Permitted Daily Exposure (PDE) may be calculated and to exemplify this, case studies of four common E&Ls are described herein, namely bisphenol-A, butylated hydroxytoluene, Irgafos® 168, and Irganox® 1010. Relevant discussion points are further explored, including the value of extractable data, how to perform route-to-route extrapolations and considerations around degradation products. By presenting PDEs for common E&L substances, the aim is to encourage consistency and harmony in approaches for deriving compound-specific limits.

Deriving harmonised permitted daily exposures (PDEs) for paracetamol (acetaminophen) CAS #: 103-90-2.

In the pharmaceutical industry, cleaning criteria are required for multipurpose manufacturing facilities. These Health Based Exposure Limits (HBELs), also called permitted daily exposures (PDEs) values, are derived from toxicological and pharmacological evaluation of the active pharmaceutical ingredients (APIs). The purpose of this publication is to show an example of how authors from different companies evaluate a generic drug, paracetamol, and discuss different approaches and relevance of the nonclinical studies for deriving PDEs. PDE limits of 25 mg/day for the oral route, and 20 mg/day for the intravenous (i.v.) and inhalation (inhal.) routes, respectively, were established herein. However, it has been already recognised that there are acceptable differences in the PDE calculations, which may be based on data accessibility, company-specific science-policy decisions or expert judgments. These differences can cause up to a 3-fold lower or higher values. If unnecessarily high factors are applied, this would result in a very conservative PDE value and unneeded additional cleaning and higher manufacturing costs. The PDE values presented are considered to be protective against adverse and pharmacological effects observed in clinical trials and in this case, a very long postmarketing period of paracetamol.

NvPOU4/Brain3 Functions as a Terminal Selector Gene in the Nervous System of the Cnidarian Nematostella vectensis.

Terminal selectors are transcription factors that control the morphological, physiological, and molecular features that characterize distinct cell types. Here, we show that, in the sea anemone Nematostella vectensis, NvPOU4 is expressed in post-mitotic cells that give rise to a diverse set of neural cell types, including cnidocytes and NvElav1-expressing neurons. Morphological analyses of NvPOU4 mutants crossed to transgenic reporter lines show that the loss of NvPOU4 does not affect the initial specification of neural cells. Transcriptomes derived from the mutants and from different neural cell populations reveal that NvPOU4 is required for the execution of the terminal differentiation program of these neural cells. These findings suggest that POU4 genes have ancient functions as terminal selectors for morphologically and functionally disparate types of neurons and they provide experimental support for the relevance of terminal selectors for understanding the evolution of cell types.

Successful management of severe coronoid process hyperplasia in a patient with mucopolysaccharidosis VI: a case report.

Mucopolysaccharidoses are a group of rare lysosomal storage diseases caused by a deficiency of enzymes, which breakdown glycosaminoglycans, with consequent dysfunction of affected tissues. Mandibular coronoid hyperplasia, with associated trismus, has been recently described as a feature of the craniofacial abnormalities seen in these patients. However, the details of the surgical and post-operative management of these patients have not been previously documented. This case describes the successful management of severe trismus from coronoid process hyperplasia in a 14-year-old male, utilising an extra-oral approach for bilateral coronoidectomies and removal of exophytic zygomatic bone, followed by immediate and long-term physiotherapy. An improvement of mandibular opening from 8 to 45 mm has been maintained at 18 months post operation.

Trans-Graft Protection Against Pierce's Disease Mediated by Transgenic Grapevine Rootstocks.

A field study showed that transgenic grapevine rootstocks can provide trans-graft-mediated protection to a wild type scion against Pierce's disease (PD) development. We individually field-tested two distinct strategies. The first expressed a chimeric antimicrobial protein (CAP) that targeted the functionality of the lipopolysaccharide (LPS) surface of Xylella fastidiosa (Xf), the causative agent of PD. The second expressed a plant polygalacturonase inhibitory protein (PGIP) that prevents PD by inhibiting breakdown of pectin present in primary cell walls. Both proteins are secreted to the apoplast and then into the xylem, where they migrate past the graft union, transiting into the xylem of the grafted scion. Transgenic Vitis vinifera cv. Thompson Seedless (TS) expressing ether CAP or PGIP were tested in the greenhouse and those lines that showed resistance to PD were grafted with wild type TS scions. Grafted grapevines were introduced into the field and tested over 7 years. Here we present data on the field evaluation of trans-graft protection using four CAP and four PGIP independent rootstock lines, compared to an untransformed rootstock. There was 30 to 95% reduction in vine mortality among CAP- and PGIP-expressing lines after three successive yearly infections with virulent Xf. Shoot tissues grafted to either CAP or PGIP transgenic rootstocks supported lower pathogen titers and showed fewer disease symptoms. Grafted plants on transgenic rootstocks also had more spring bud break following infection, more shoots, and more vigorous growth compared to those grafted to wild type rootstocks. No yield penalty was observed in the transgenic lines and some PGIP-expressing vines had enhanced yield potential. Trans-graft protection is an efficient way to protect grape scions against PD while preserving their valuable varietal genotypes and clonal properties.