Identification of in vivo roles of ErbB4-JMa and its direct nuclear signaling using a novel isoform-specific knock out mouse.

Like all receptor tyrosine kinases (RTKs), ErbB4 signals through a canonical signaling involving phosphorylation cascades. However, ErbB4 can also signal through a non-canonical mechanism whereby the intracellular domain is released into the cytoplasm by regulated intramembrane proteolysis (RIP) and translocates to the nucleus where it regulates transcription. These different signaling mechanisms depend on the generation of alternative spliced isoforms, a RIP cleavable ErbB4-JMa and an uncleavable ErbB4-JMb. Non-canonical signaling by ErbB4-JMa has been implicated in the regulation of brain, heart, mammary gland, lung, and immune cell development. However, most studies on non-canonical ErbB4 signaling have been performed in vitro due to the lack of an adequate mouse model. We created an ErbB4-JMa specific knock out mouse and demonstrate that RIP-dependent, non-canonical signaling by ErbB4-JMa is required for the regulation of GFAP expression during cortical development. We also show that ErbB4-JMa signaling is not required for the development of the heart, mammary glands, sensory ganglia. Furthermore, we identify genes whose expression during cortical development is regulated by ErbB4, and show that the expression of three of them, CRYM and DBi, depend on ErbB4-JMa whereas WDFY1 relies on ErbB4-JMb. Thus, we provide the first animal model to directly study the roles of ErbB4-JMa and non-canonical ErbB4 signaling in vivo.

Poly (ADP-Ribose) Polymerase 1 Regulates Cajal-Retzius Cell Development and Neural Precursor Cell Adhesion.

Poly (ADP-ribose) polymerase 1 (PARP1) is a ubiquitously expressed enzyme that regulates DNA damage repair, cell death, inflammation, and transcription. PARP1 functions by adding ADP-ribose polymers (PAR) to proteins including itself, using NAD+ as a donor. This post-translational modification known as PARylation results in changes in the activity of PARP1 and its substrate proteins and has been linked to the pathogenesis of various neurological diseases. PARP1 KO mice display schizophrenia-like behaviors, have impaired memory formation, and have defects in neuronal proliferation and survival, while mutations in genes that affect PARylation have been associated with intellectual disability, psychosis, neurodegeneration, and stroke in humans. Yet, the roles of PARP1 in brain development have not been extensively studied. We now find that loss of PARP1 leads to defects in brain development and increased neuronal density at birth. We further demonstrate that PARP1 loss increases the expression levels of genes associated with neuronal migration and adhesion in the E15.5 cerebral cortex, including Reln. This correlates with an increased number of Cajal-Retzius (CR) cells in vivo and in cultures of embryonic neural progenitor cells (NPCs) derived from the PARP1 KO cortex. Furthermore, PARP1 loss leads to increased NPC adhesion to N-cadherin, like that induced by experimental exposure to Reelin. Taken together, these results uncover a novel role for PARP1 in brain development, i.e., regulation of CR cells, neuronal density, and cell adhesion.

Rapid molecular detection of macrolide resistance.

BACKGROUND: Emerging antimicrobial resistance is a significant threat to human health. However, methods for rapidly diagnosing antimicrobial resistance generally require multi-day culture-based assays. Macrolide efflux gene A, mef(A), provides resistance against erythromycin and azithromycin and is known to be laterally transferred among a wide range of bacterial species. METHODS: We use Recombinase Polymerase Assay (RPA) to detect the antimicrobial resistance gene mef(A) from raw lysates without nucleic acid purification. To validate these results we performed broth dilution assays to assess antimicrobial resistance to erythromycin and ampicillin (a negative control). RESULTS: We validate the detection of mef(A) in raw lysates of Streptococcus pyogenes, S. pneumoniae, S. salivarius, and Enterococcus faecium bacterial lysates within 7-10 min of assay time. We show that detection of mef(A) accurately predicts real antimicrobial resistance assessed by traditional culture methods, and that the assay is robust to high levels of spiked-in non-specific nucleic acid contaminant. The assay was unaffected by single-nucleotide polymorphisms within divergent mef(A) gene sequences, strengthening its utility as a robust diagnostic tool. CONCLUSIONS: This finding opens the door to implementation of rapid genomic diagnostics in a clinical setting, while providing researchers a rapid, cost-effective tool to track antibiotic resistance in both pathogens and commensal strains.

Novel Sequence Discovery by Subtractive Genomics.

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a larger genomic context. Subtractive genomics enables a researcher to isolate a target sequence of interest (T) by comprehensive sequencing and subtracting out known genetic elements (reference, R). The method can be used to identify novel sequences such as mitochondria, chloroplasts, viruses, or germline restricted chromosomes, and is particularly useful when T cannot be easily isolated from R. Beginning with the comprehensive genomic data (R + T), the method uses Basic Local Alignment Search Tool (BLAST) against a reference sequence, or sequences, to remove the matching known sequences (R), leaving behind the target (T). For subtraction to work best, R should be a relatively complete draft that is missing T. Since sequences remaining after subtraction are tested through quantitative Polymerase Chain Reaction (qPCR), R does not need to be complete for the method to work. Here we link computational steps with experimental steps into a cycle that can be iterated as needed, sequentially removing multiple reference sequences and refining the search for T. The advantage of subtractive genomics is that a completely novel target sequence can be identified even in cases in which physical purification is difficult, impossible, or expensive. A drawback of the method is finding a suitable reference for subtraction and obtaining T-positive and negative samples for qPCR testing. We describe our implementation of the method in the identification of the first gene from the germline-restricted chromosome of zebra finch. In that case computational filtering involved three references (R), sequentially removed over three cycles: an incomplete genomic assembly, raw genomic data, and transcriptomic data.

Discovery of the First Germline-Restricted Gene by Subtractive Transcriptomic Analysis in the Zebra Finch, Taeniopygia guttata.

Developmentally programmed genome rearrangements are rare in vertebrates, but have been reported in scattered lineages including the bandicoot, hagfish, lamprey, and zebra finch (Taeniopygia guttata) [1]. In the finch, a well-studied animal model for neuroendocrinology and vocal learning [2], one such programmed genome rearrangement involves a germline-restricted chromosome, or GRC, which is found in germlines of both sexes but eliminated from mature sperm [3, 4]. Transmitted only through the oocyte, it displays uniparental female-driven inheritance, and early in embryonic development is apparently eliminated from all somatic tissue in both sexes [3, 4]. The GRC comprises the longest finch chromosome at over 120 million base pairs [3], and previously the only known GRC-derived sequence was repetitive and non-coding [5]. Because the zebra finch genome project was sourced from male muscle (somatic) tissue [6], the remaining genomic sequence and protein-coding content of the GRC remain unknown. Here we report the first protein-coding gene from the GRC: a member of the α-soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein (α-SNAP) family hitherto missing from zebra finch gene annotations. In addition to the GRC-encoded α-SNAP, we find an additional paralogous α-SNAP residing in the somatic genome (a somatolog)-making the zebra finch the first example in which α-SNAP is not a single-copy gene. We show divergent, sex-biased expression for the paralogs and also that positive selection is detectable across the bird α-SNAP lineage, including the GRC-encoded α-SNAP. This study presents the identification and evolutionary characterization of the first protein-coding GRC gene in any organism.

Viral-mediated Ntf3 overexpression disrupts innervation and hearing in nondeafened guinea pig cochleae.

Synaptopathy in the cochlea occurs when the connection between inner hair cells and the auditory nerve is disrupted, leading to impaired hearing and nerve degeneration. Experiments using transgenic mice have shown that overexpression of NT3 by supporting cells repairs synaptopathy caused by overstimulation. To accomplish such therapy in the clinical setting, it would be necessary to activate the neurotrophin receptor on auditory neurons by other means. Here we test the outcome of NT3 overexpression using viral-mediated gene transfer into the perilymph versus the endolymph of the normal guinea pig cochlea. We inoculated two different Ntf3 viral vectors, adenovirus (Adv) or adeno-associated virus (AAV) into the perilymph, to facilitate transgene expression in the mesothelial cells and cochlear duct epithelium, respectively. We assessed outcomes by comparing Auditory brainstem response (ABR) thresholds prior to that at baseline to thresholds at 1 and 3 weeks after inoculation, and then performed histologic evaluation of hair cells, nerve endings, and synaptic ribbons. We observed hearing threshold shifts as well as disorganization of peripheral nerve endings and disruption of synaptic connections between inner hair cells and peripheral nerve endings with both vectors. The data suggest that elevation of NT3 levels in the cochlear fluids can disrupt innervation and degrade hearing.