Endoplasmic Reticulum Calcium Mediates Drosophila Wing Development.

Background: The temporal dynamics of morphogen presentation impacts transcriptional responses and tissue patterning. However, the mechanisms controlling morphogen release are far from clear. We found that inwardly rectifying potassium (Irk) channels regulate endogenous transient increases in intracellular calcium and bone morphogenetic protein (BMP/Dpp) release for Drosophila wing development. Inhibition of Irk channels reduces BMP/Dpp signaling, and ultimately disrupts wing morphology. Ion channels impact development of several tissues and organisms in which BMP signaling is essential. In neurons and pancreatic beta cells, Irk channels modulate membrane potential to affect intracellular Ca++ to control secretion of neurotransmitters and insulin. Based on Irk activity in neurons, we hypothesized that electrical activity controls endoplasmic reticulum (ER) Ca++ release into the cytoplasm to regulate the release of BMP. Materials and Methods: To test this hypothesis, we reduced expression of four proteins that control ER calcium, Stromal interaction molecule 1 (Stim), Calcium release-activated calcium channel protein 1 (Orai), SarcoEndoplasmic Reticulum Calcium ATPase (SERCA), small conductance calcium-activated potassium channel (SK), and Bestrophin 2 (Best2) using RNAi and documented wing phenotypes. We use live imaging to study calcium and Dpp release within pupal wings and larval wing discs. Additionally, we employed immunohistochemistry to characterize Small Mothers Against Decapentaplegic (SMAD) phosphorylation downstream of the BMP/Dpp pathway following RNAi knockdown. Results: We found that reduced Stim and SERCA function decreases amplitude and frequency of endogenous calcium transients in the wing disc and reduced BMP/Dpp release. Conclusion: Our results suggest control of ER calcium homeostasis is required for BMP/Dpp release, and Drosophila wing development.

Fetal cannabidiol (CBD) exposure alters thermal pain sensitivity, problem-solving, and prefrontal cortex excitability.

Thousands of people suffer from nausea with pregnancy each year. Nausea can be alleviated with cannabidiol (CBD), a primary component of cannabis that is widely available. However, it is unknown how fetal CBD exposure affects embryonic development and postnatal outcomes. CBD binds and activates receptors that are expressed in the fetal brain and are important for brain development, including serotonin receptors (5HT1A), voltage-gated potassium (Kv)7 receptors, and the transient potential vanilloid 1 receptor (TRPV1). Excessive activation of each of these receptors can disrupt neurodevelopment. Here, we test the hypothesis that fetal CBD exposure in mice alters offspring neurodevelopment and postnatal behavior. We administered 50 mg/kg CBD in sunflower oil or sunflower oil alone to pregnant mice from embryonic day 5 through birth. We show that fetal CBD exposure sensitizes adult male offspring to thermal pain through TRPV1. We show that fetal CBD exposure decreases problem-solving behaviors in female CBD-exposed offspring. We demonstrate that fetal CBD exposure increases the minimum current required to elicit action potentials and decreases the number of action potentials in female offspring layer 2/3 prefrontal cortex (PFC) pyramidal neurons. Fetal CBD exposure reduces the amplitude of glutamate uncaging-evoked excitatory post-synaptic currents, consistent with CBD-exposed female problem-solving behavior deficits. Combined, these data show that fetal CBD exposure disrupts neurodevelopment and postnatal behavior in a sex specific manner.

Intrauterine exposure to nicotine through maternal vaping disrupts embryonic lung and skeletal development via the Kcnj2 potassium channel.

Smoking cigarettes during pregnancy is associated with adverse effects on infants including low birth weight, defective lung development, and skeletal abnormalities. Pregnant women are increasingly turning to vaping [use of electronic (e)-cigarettes] as a perceived safer alternative to cigarettes. However, nicotine disrupts fetal development, suggesting that like cigarette smoking, nicotine vaping may be detrimental to the fetus. To test the impact of maternal vaping on fetal lung and skeletal development in mice, pregnant dams were exposed to e-cigarette vapor throughout gestation. At embryonic day (E)18.5, vape exposed litter sizes were reduced, and some embryos exhibited growth restriction compared to air exposed controls. Fetal lungs were collected for histology and whole transcriptome sequencing. Maternally nicotine vaped embryos exhibited histological and transcriptional changes consistent with impaired distal lung development. Embryonic lung gene expression changes mimicked transcriptional changes observed in adult mouse lungs exposed to cigarette smoke, suggesting that the developmental defects may be due to direct nicotine exposure. Fetal skeletons were analyzed for craniofacial and long bone lengths. Nicotine directly binds and inhibits the Kcnj2 potassium channel which is important for bone development. The length of the maxilla, palatal shelves, humerus, and femur were reduced in vaped embryos, which was further exacerbated by loss of one copy of the Kcnj2 gene. Nicotine vapor exposed Kcnj2KO/+ embryos also had significantly lower birth weights than unexposed animals of either genotype. Kcnj2 mutants had severely defective lungs with and without vape exposure, suggesting that potassium channels may be broadly involved in mediating the detrimental developmental effects of nicotine vaping. These data indicate that intrauterine nicotine exposure disrupts fetal lung and skeletal development likely through inhibition of Kcnj2.

Engineering Adenoviral Vectors with Improved GBM Selectivity.

Glioblastoma (GBM) is the most common and aggressive adult brain cancer with an average survival rate of around 15 months in patients receiving standard treatment. Oncolytic adenovirus expressing therapeutic transgenes represent a promising alternative treatment for GBM. Of the many human adenoviral serotypes described to date, adenovirus 5 (HAdV-C5) has been the most utilised clinically and experimentally. However, the use of Ad5 as an anti-cancer agent may be hampered by naturally high seroprevalence rates to HAdV-C5 coupled with the infection of healthy cells via native receptors. To explore whether alternative natural adenoviral tropisms are better suited to GBM therapeutics, we pseudotyped an HAdV-C5-based platform using the fibre knob protein from alternative serotypes. We demonstrate that the adenoviral entry receptor coxsackie, adenovirus receptor (CAR) and CD46 are highly expressed by both GBM and healthy brain tissue, whereas Desmoglein 2 (DSG2) is expressed at a low level in GBM. We demonstrate that adenoviral pseudotypes, engaging CAR, CD46 and DSG2, effectively transduce GBM cells. However, the presence of these receptors on non-transformed cells presents the possibility of off-target effects and therapeutic transgene expression in healthy cells. To enhance the specificity of transgene expression to GBM, we assessed the potential for tumour-specific promoters hTERT and survivin to drive reporter gene expression selectively in GBM cell lines. We demonstrate tight GBM-specific transgene expression using these constructs, indicating that the combination of pseudotyping and tumour-specific promoter approaches may enable the development of efficacious therapies better suited to GBM.

Associations between Prenatal and Postnatal Exposure to Cannabis with Cognition and Behavior at Age 5 Years: The Healthy Start Study.

BACKGROUND: Prenatal exposure to cannabis may influence childhood cognition and behavior, but the epidemiologic evidence is mixed. Even less is known about the potential impact of secondhand exposure to cannabis during early childhood. OBJECTIVE: This study sought to assess whether prenatal and/or postnatal exposure to cannabis was associated with childhood cognition and behavior. STUDY DESIGN: This sub-study included a convenience sample of 81 mother-child pairs from a Colorado-based cohort. Seven common cannabinoids (including delta 9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD)) and their metabolites were measured in maternal urine collected mid-gestation and child urine collected at age 5 years. Prenatal and postnatal exposure to cannabis was dichotomized as exposed (detection of any cannabinoid) and not exposed. Generalized linear models examined the associations between prenatal or postnatal exposure to cannabis with the NIH Toolbox and Child Behavior Checklist T-scores at age 5 years. RESULTS: In this study, 7% (n = 6) of the children had prenatal exposure to cannabis and 12% (n = 10) had postnatal exposure to cannabis, with two children experiencing this exposure at both time points. The most common cannabinoid detected in pregnancy was Δ9-THC, whereas the most common cannabinoid detected in childhood was CBD. Postnatal exposure to cannabis was associated with more aggressive behavior (ß: 3.2; 95% CI: 0.5, 5.9), attention deficit/hyperactivity problems (ß: 8.0; 95% CI: 2.2, 13.7), and oppositional/defiant behaviors (ß: 3.2; 95% CI: 0.2, 6.3), as well as less cognitive flexibility (ß: -15.6; 95% CI: -30.0, -1.2) and weaker receptive language (ß: -9.7; 95% CI: -19.2, -0.3). By contrast, prenatal exposure to cannabis was associated with fewer internalizing behaviors (mean difference: -10.2; 95% CI: -20.3, -0.2) and fewer somatic complaints (mean difference: -5.2, 95% CI: -9.8, -0.6). CONCLUSIONS: Our study suggests that postnatal exposure to cannabis is associated with more behavioral and cognitive problems among 5-year-old children, independent of prenatal and postnatal exposure to tobacco. The potential risks of cannabis use (including smoking and vaping) during pregnancy and around young children should be more widely communicated to parents.

Single cell sequencing of the mouse anterior palate reveals mesenchymal heterogeneity.

BACKGROUND: Cleft palate is one of the most prevalent birth defects. Mice are useful for studying palate development because of their morphological and genetic similarities to humans. In mice, palate development occurs between embryonic days (E)11.5 to 15.5. Single cell transcriptional profiles of palate cell populations have been a valuable resource for the craniofacial research community, but we lack a single cell transcriptional profile for anterior palate at E13.5, at the transition from proliferation to shelf elevation. RESULTS: A detailed single cell RNA sequencing analysis reveals heterogeneity in expression profiles of the cell populations of the E13.5 anterior palate. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA FISH) reveals epithelial populations segregate into layers. Mesenchymal populations spatially segregate into four domains. One of these mesenchymal populations expresses ligands and receptors distinct from the rest of the mesenchyme, suggesting that these cells have a unique function. RNA velocity analysis shows two terminal cell states that contribute to either the proximal or distal palatal regions emerge from a single progenitor pool. CONCLUSION: This single cell resolution expression data and detailed analysis from E13.5 anterior palate provides a powerful resource for mechanistic insight into secondary palate morphogenesis for the craniofacial research community.

Broad sialic acid usage amongst species D human adenovirus.

Human adenoviruses (HAdV) are widespread pathogens causing usually mild infections. The Species D (HAdV-D) cause gastrointestinal tract infections and epidemic keratoconjunctivitis (EKC). Despite being significant pathogens, knowledge around HAdV-D mechanism of cell infection is lacking. Sialic acid (SA) usage has been proposed as a cell infection mechanism for EKC causing HAdV-D. Here we highlight an important role for SA engagement by many HAdV-D. We provide apo state crystal structures of 7 previously undetermined HAdV-D fiber-knob proteins, and structures of HAdV-D25, D29, D30 and D53 fiber-knob proteins in complex with SA. Biologically, we demonstrate that removal of cell surface SA reduced infectivity of HAdV-C5 vectors pseudotyped with HAdV-D fiber-knob proteins, whilst engagement of the classical HAdV receptor CAR was variable. Our data indicates variable usage of SA and CAR across HAdV-D. Better defining these interactions will enable improved development of antivirals and engineering of the viruses into refined therapeutic vectors.

Development of a low-seroprevalence, αvß6 integrin-selective virotherapy based on human adenovirus type 10.

Oncolytic virotherapies (OV) hold immense clinical potential. OV based on human adenoviruses (HAdV) derived from HAdV with naturally low rates of pre-existing immunity will be beneficial for future clinical translation. We generated a low-seroprevalence HAdV-D10 serotype vector incorporating an αvß6 integrin-selective peptide, A20, to target αvß6-positive tumor cell types. HAdV-D10 has limited natural tropism. Structural and biological studies of HAdV-D10 knob protein highlighted low-affinity engagement with native adenoviral receptors CAR and sialic acid. HAdV-D10 fails to engage blood coagulation factor X, potentially eliminating "off-target" hepatic sequestration in vivo. We engineered an A20 peptide that selectively binds αvß6 integrin into the DG loop of HAdV-D10 fiber knob. Assays in αvß6+ cancer cell lines demonstrated significantly increased transduction mediated by αvß6-targeted variants compared with controls, confirmed microscopically. HAdV-D10.A20 resisted neutralization by neutralizing HAdV-C5 sera. Systemic delivery of HAdV-D10.A20 resulted in significantly increased GFP expression in BT20 tumors. Replication-competent HAdV-D10.A20 demonstrated αvß6 integrin-selective cell killing in vitro and in vivo. HAdV-D10 possesses characteristics of a promising virotherapy, combining low seroprevalence, weak receptor interactions, and reduced off-target uptake. Incorporation of an αvß6 integrin-selective peptide resulted in HAdV-D10.A20, with significant potential for clinical translation.

Mechanisms Underlying Influence of Bioelectricity in Development.

To execute the intricate process of development, cells coordinate across tissues and organs to determine where each cell divides and differentiates. This coordination requires complex communication between cells. Growing evidence suggests that bioelectrical signals controlled via ion channels contribute to cell communication during development. Ion channels collectively regulate the transmembrane potential of cells, and their function plays a conserved role in the development of organisms from flies to humans. Spontaneous calcium oscillations can be found in nearly every cell type and tissue, and disruption of these oscillations leads to defects in development. However, the mechanism by which bioelectricity regulates development is still unclear. Ion channels play essential roles in the processes of cell death, proliferation, migration, and in each of the major canonical developmental signaling pathways. Previous reviews focus on evidence for one potential mechanism by which bioelectricity affects morphogenesis, but there is evidence that supports multiple different mechanisms which are not mutually exclusive. Evidence supports bioelectricity contributing to development through multiple different mechanisms. Here, we review evidence for the importance of bioelectricity in morphogenesis and provide a comprehensive review of the evidence for several potential mechanisms by which ion channels may act in developmental processes.

ChAdOx1 interacts with CAR and PF4 with implications for thrombosis with thrombocytopenia syndrome.

Vaccines derived from chimpanzee adenovirus Y25 (ChAdOx1), human adenovirus type 26 (HAdV-D26), and human adenovirus type 5 (HAdV-C5) are critical in combatting the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic. As part of the largest vaccination campaign in history, ultrarare side effects not seen in phase 3 trials, including thrombosis with thrombocytopenia syndrome (TTS), a rare condition resembling heparin-induced thrombocytopenia (HIT), have been observed. This study demonstrates that all three adenoviruses deployed as vaccination vectors versus SARS-CoV-2 bind to platelet factor 4 (PF4), a protein implicated in the pathogenesis of HIT. We have determined the structure of the ChAdOx1 viral vector and used it in state-of-the-art computational simulations to demonstrate an electrostatic interaction mechanism with PF4, which was confirmed experimentally by surface plasmon resonance. These data confirm that PF4 is capable of forming stable complexes with clinically relevant adenoviruses, an important step in unraveling the mechanisms underlying TTS.

In Vitro and In Vivo Evaluation of Human Adenovirus Type 49 as a Vector for Therapeutic Applications.

The human adenovirus phylogenetic tree is split across seven species (A-G). Species D adenoviruses offer potential advantages for gene therapy applications, with low rates of pre-existing immunity detected across screened populations. However, many aspects of the basic virology of species D-such as their cellular tropism, receptor usage, and in vivo biodistribution profile-remain unknown. Here, we have characterized human adenovirus type 49 (HAdV-D49)-a relatively understudied species D member. We report that HAdV-D49 does not appear to use a single pathway to gain cell entry, but appears able to interact with various surface molecules for entry. As such, HAdV-D49 can transduce a broad range of cell types in vitro, with variable engagement of blood coagulation FX. Interestingly, when comparing in vivo biodistribution to adenovirus type 5, HAdV-D49 vectors show reduced liver targeting, whilst maintaining transduction of lung and spleen. Overall, this presents HAdV-D49 as a robust viral vector platform for ex vivo manipulation of human cells, and for in vivo applications where the therapeutic goal is to target the lung or gain access to immune cells in the spleen, whilst avoiding liver interactions, such as intravascular vaccine applications.

Bridging the Gap: The Importance of TUBA1A α-Tubulin in Forming Midline Commissures.

Developing neurons undergo dramatic morphological changes to appropriately migrate and extend axons to make synaptic connections. The microtubule cytoskeleton, made of α/ß-tubulin dimers, drives neurite outgrowth, promotes neuronal growth cone responses, and facilitates intracellular transport of critical cargoes during neurodevelopment. TUBA1A constitutes the majority of α-tubulin in the developing brain and mutations to TUBA1A in humans cause severe brain malformations accompanied by varying neurological defects, collectively termed tubulinopathies. Studies of TUBA1A function in mammalian cells have been limited by the presence of multiple genes encoding highly similar tubulin proteins, which leads to α-tubulin antibody promiscuity and makes genetic manipulation challenging. Here, we test mutant tubulin levels and assembly activity and analyze the impact of TUBA1A reduction on growth cone composition, neurite extension, and commissural axon architecture during brain development. We present a novel tagging method for studying and manipulating TUBA1A in cells without impairing tubulin function. Using this tool, we show that a TUBA1A loss-of-function mutation TUBA1A N102D (TUBA1A ND ), reduces TUBA1A protein levels and prevents incorporation of TUBA1A into microtubule polymers. Reduced Tuba1a α-tubulin in heterozygous Tuba1a ND/+ mice leads to grossly normal brain formation except a significant impact on axon extension and impaired formation of forebrain commissures. Neurons with reduced Tuba1a as a result of the Tuba1a ND mutation exhibit slower neuron outgrowth compared to controls. Neurons deficient in Tuba1a failed to localize microtubule associated protein-1b (Map1b) to the developing growth cone, likely impacting stabilization of microtubules. Overall, we show that reduced Tuba1a is sufficient to support neuronal migration and cortex development but not commissure formation, and provide mechanistic insight as to how TUBA1A tunes microtubule function to support neurodevelopment.

The Fiber Knob Protein of Human Adenovirus Type 49 Mediates Highly Efficient and Promiscuous Infection of Cancer Cell Lines Using a Novel Cell Entry Mechanism.

The human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of preexisting immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases, where they induce robust immunity in preclinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied. Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids from HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, while potential interaction with coxsackievirus and adenovirus receptor (CAR) is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, esophageal, and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumor types.IMPORTANCE Adenoviruses are powerful tools experimentally and clinically. To maximize efficacy, the development of serotypes with low preexisting levels of immunity in the population is desirable. Consequently, attention has focused on those derived from species D, which have proven robust vaccine platforms. This widespread usage is despite limited knowledge in their basic biology and cellular tropism. We investigated the tropism of HAdV-D49, demonstrating that it uses a novel cell entry mechanism that bypasses all known HAdV receptors. We demonstrate, biologically, that a pseudotyped HAdV-C5/D49K vector efficiently transduces a wide range of cell lines, including those presenting no known adenovirus receptor. Structural investigation suggests that this broad tropism is the result of a highly basic electrostatic surface potential, since a homologous pseudotyped vector with a more acidic surface potential, HAdV-C5/D30K, does not display a similar pantropism. Therefore, HAdV-C5/D49K may form a powerful vector for therapeutic applications capable of infecting difficult to transduce cells.

Hitting the Target but Missing the Point: Recent Progress towards Adenovirus-Based Precision Virotherapies.

More people are surviving longer with cancer. Whilst this can be partially attributed to advances in early detection of cancers, there is little doubt that the improvement in survival statistics is also due to the expansion in the spectrum of treatments available for efficacious treatment. Transformative amongst those are immunotherapies, which have proven effective agents for treating immunogenic forms of cancer, although immunologically "cold" tumour types remain refractive. Oncolytic viruses, such as those based on adenovirus, have great potential as anti-cancer agents and have seen a resurgence of interest in recent years. Amongst their many advantages is their ability to induce immunogenic cell death (ICD) of infected tumour cells, thus providing the alluring potential to synergise with immunotherapies by turning immunologically "cold" tumours "hot". Additionally, enhanced immune mediated cell killing can be promoted through the local overexpression of immunological transgenes, encoded from within the engineered viral genome. To achieve this full potential requires the development of refined, tumour selective "precision virotherapies" that are extensively engineered to prevent off-target up take via native routes of infection and targeted to infect and replicate uniquely within malignantly transformed cells. Here, we review the latest advances towards this holy grail within the adenoviral field.

Ion Channel Contributions to Morphological Development: Insights From the Role of Kir2.1 in Bone Development.

The role of ion channels in neurons and muscles has been well characterized. However, recent work has demonstrated both the presence and necessity of ion channels in diverse cell types for morphological development. For example, mutations that disrupt ion channels give rise to abnormal structural development in species of flies, frogs, fish, mice, and humans. Furthermore, medications and recreational drugs that target ion channels are associated with higher incidence of birth defects in humans. In this review we establish the effects of several teratogens on development including epilepsy treatment drugs (topiramate, valproate, ethosuximide, phenobarbital, phenytoin, and carbamazepine), nicotine, heat, and cannabinoids. We then propose potential links between these teratogenic agents and ion channels with mechanistic insights from model organisms. Finally, we talk about the role of a particular ion channel, Kir2.1, in the formation and development of bone as an example of how ion channels can be used to uncover important processes in morphogenesis. Because ion channels are common targets of many currently used medications, understanding how ion channels impact morphological development will be important for prevention of birth defects. It is becoming increasingly clear that ion channels have functional roles outside of tissues that have been classically considered excitable.

Reduced TUBA1A Tubulin Causes Defects in Trafficking and Impaired Adult Motor Behavior.

Newly born neurons express high levels of TUBA1A α-tubulin to assemble microtubules for neurite extension and to provide tracks for intracellular transport. In the adult brain, Tuba1a expression decreases dramatically. A mouse that harbors a loss-of-function mutation in the gene encoding TUBA1A (Tuba1aND/+ ) allows us to ask whether TUBA1A is important for the function of mature neurons. α-Tubulin levels are about half of wild type in juvenile Tuba1aND/+ brains, but are close to normal in older animals. In postnatal day (P)0 cultured neurons, reduced TUBA1A allows for assembly of less microtubules in axons resulting in more pausing during organelle trafficking. While Tuba1aND/+ mouse behavior is indistinguishable from wild-type siblings at weaning, Tuba1aND/+ mice develop adult-onset ataxia. Neurons important for motor function in Tuba1aND/+ remain indistinguishable from wild-type with respect to morphology and number and display no evidence of axon degeneration. Tuba1aND/+ neuromuscular junction (NMJ) synapses are the same size as wild-type before the onset of ataxia, but are reduced in size in older animals. Together, these data indicate that the TUBA1A-rich microtubule tracks that are assembled during development are essential for mature neuron function and maintenance of synapses over time.

Identification of folate receptor α (FRα) binding oligopeptides and their evaluation for targeted virotherapy applications.

Oncolytic virotherapies (OV) based on human adenoviral (HAdV) vectors hold significant promise for the treatment of advanced ovarian cancers where local, intraperitoneal delivery to tumour metastases is feasible, bypassing many complexities associated with intravascular delivery. The efficacy of HAdV-C5-based OV is hampered by a lack of tumour selectivity, where the primary receptor, hCAR, is commonly downregulated during malignant transformation. Conversely, folate receptor alpha (FRα) is highly expressed on ovarian cancer cells, providing a compelling target for tumour selective delivery of virotherapies. Here, we identify high-affinity FRα-binding oligopeptides for genetic incorporation into HAdV-C5 vectors. Biopanning identified a 12-mer linear peptide, DWSSWVYRDPQT, and two 7-mer cysteine-constrained peptides, CIGNSNTLC and CTVRTSAEC that bound FRα in the context of the phage particle. Synthesised lead peptide, CTVRTSAEC, bound specifically to FRα and could be competitively inhibited with folic acid. To assess the capacity of the elucidated FRα-binding oligopeptides to target OV to FRα, we genetically incorporated the peptides into the HAdV-C5 fiber-knob HI loop including in vectors genetically ablated for hCAR interactions. Unfortunately, the recombinant vectors failed to efficiently target transduction via FRα due to defective intracellular trafficking following entry via FRα, indicating that whilst the peptides identified may have potential for applications for targeted drug delivery, they require additional refinement for targeted virotherapy applications.

Tubulin mutations in brain development disorders: Why haploinsufficiency does not explain TUBA1A tubulinopathies.

The neuronal cytoskeleton performs incredible feats during nervous system development. Extension of neuronal processes, migration, and synapse formation rely on the proper regulation of microtubules. Mutations that disrupt the primary α-tubulin expressed during brain development, TUBA1A, are associated with a spectrum of human brain malformations. One model posits that TUBA1A mutations lead to a reduction in tubulin subunits available for microtubule polymerization, which represents a haploinsufficiency mechanism. We propose an alternative model for the majority of tubulinopathy mutations, in which the mutant tubulin polymerizes into the microtubule lattice to dominantly "poison" microtubule function. Nine distinct α-tubulin and ten ß-tubulin genes have been identified in the human genome. These genes encode similar tubulin proteins, called isotypes. Multiple tubulin isotypes may partially compensate for heterozygous deletion of a tubulin gene, but may not overcome the disruption caused by missense mutations that dominantly alter microtubule function. Here, we describe disorders attributed to haploinsufficiency versus dominant negative mechanisms to demonstrate the hallmark features of each disorder. We summarize literature on mouse models that represent both knockout and point mutants in tubulin genes, with an emphasis on how these mutations might provide insight into the nature of tubulinopathy patient mutations. Finally, we present data from a panel of TUBA1A tubulinopathy mutations generated in yeast α-tubulin that demonstrate that α-tubulin mutants can incorporate into the microtubule network and support viability of yeast growth. This perspective on tubulinopathy mutations draws on previous studies and additional data to provide a fresh perspective on how TUBA1A mutations disrupt neurodevelopment.

Imaging Dpp Release from a Drosophila Wing Disc.

The transforming Growth Factor-beta (TGF-ß) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-ß superfamily includes TGF-ß, bone morphogenetic protein (BMPs), Activins, Growth and Differentiation Factors, and Nodals. It has long been known that the amount of ligand exposed to cells is important for its effects. It was thought that long-range concentration gradients set up embryonic pattern. However, recently it has become clear that the timing of exposure to these ligands is also important for their downstream transcriptional consequences. A TGF-ß superfamily ligand cannot have a developmental consequence until it is released from the cell in which it was produced. Until recently, it was difficult to determine when these ligands were released from cells. Here we show how to measure the release of a Drosophila BMP called Decapentaplegic (Dpp) from the cells of the wing primordium or wing disc. This method could be modified for other systems or signaling ligands.