Mammalian Types Research Articles

Receptor complex and signalling pathway of the two type II IFNs, IFN-γ and IFN-γrel in mandarin fish or the so-called Chinese perch Siniperca chuatsi

IFN-γ, as the sole member of mammalian type II IFN, is a multifunctional cytokine which exerts its effects through two distinct IFN-γ receptors, IFNGR1 and IFNGR2. However, in teleost fish, another IFN-γ homologous gene, namely IFN-γ related gene (IFN-γrel), has been identified. Although IFN-γ and IFN-γrel genes have been described in some fish species, many important aspects remain poorly understood in relation with their signalling and function. In the present study, IFN-γ and IFN-γrel, as well as their receptors, cytokine receptor family B (CRFB) 17, CRFB13, two of which are homologous to IFNGR1 in mammals, and CRFB6, homolomous to IFNGR2, have been characterized in mandarin fish, Siniperca chuatsi. It was revealed that the two type IFN members exhibit antiviral activity, and IFN-γ transduces downstream signalling through CRFB13 and CRFB6, while IFN-γrel interacts with CRFB17 to activate downstream signalling. Moreover, IFN-γ and IFN-γrel have been shown to exert antiviral biological activity in a STAT1-dependent manner. Intracellular domain analysis of CRFB17 and CRFB13 demonstrated that the Y386 tyrosine residue of CRFB13 is required for the activation of the IFN-γ-mediated biologic response, and the Y324 and Y370 residues in CRFB17 are required to activate IFN-γrel signalling.

Host range of mammalian orthoreovirus type 3 widening to alpine chamois

Mammalian orthoreoviruses (MRV) type 3 have been recently identified in human and several animal hosts, highlighting the apparent lack of species barriers. Here we report the identification and genetic characterization of MRVs strains in alpine chamois, one of the most abundant wild ungulate in the Alps. Serological survey was also performed by MRV neutralization test in chamois population during five consecutive years (2008-2012). Three novel MRVs were isolated on cell culture from chamois lung tissues. No respiratory or other clinical symptoms neither lung macroscopic lesions were observed in the chamois population. MRV strains were classified as MRV-3 within the lineage III, based on S1 phylogeny, and were closely related to Italian strains identified in dog, bat and diarrheic pig. The full genome sequence was obtained by next-generation sequencing and phylogenetic analyses showed that other segments were more similar to MRVs of different geographic locations, serotypes and hosts, including human, highlighting genome reassortment and lack of host specific barriers. By using serum neutralization test, a high prevalence of MRV-3 antibodies was observed in chamois population throughout the monitored period, showing an endemic level of infection and suggesting a self-maintenance of MRV and/or a continuous spill-over of infection from other animal species.

Identification and expression analysis of IL-4/13 receptors in grass carp Ctenopharyngodon idella

Interleukin (IL)-4 and IL-13 are T helper 2 (Th2) cytokines with pleiotropic functions. IL-4 interacts with two receptors consisting of IL-4Rα/γ chain receptor (γC) and IL-4Rα/IL-13Rα1. In contrast, IL-13 binds to IL-13Rα2 but also shares the receptor complex containing IL-4Rα/IL-13Rα1. In fish, two IL-4/13 homologs have been identified but their phylogenetic relationships with IL-4 and IL-13 are ambiguous. In this study, we identified six putative IL-4/13 receptor homologs in grass carp, including γC1, γC2, IL-4Rα1, IL-13Rα1, IL-13Rα2 and a soluble form of IL-4Rα2. Comparative sequence analyses revealed that these receptors possess conserved characteristic domains and the genes encoding them share conserved gene synteny with their human counterparts. All six receptors contain a cytokine binding homology domain (CHD) and two fibronectin type Ⅲ (FNⅢ) like domains, with IL-13Rα1 and IL-13Rα2 harbouring an extra Ig-like domain preceding the CHD domain. Interestingly, grass carp IL-13Rα1 and IL-13Rα2 lack the characteristic WSXWS motif, a typical feature of mammalian type I cytokine receptors. The IL-4/13 receptor genes are differentially expressed in tissues and primary leukocytes of head kidney and can be modulated by Flavobacterium cloumnare (F. cloumnare), suggesting they are involved in immune response against F. cloumnare infection.

Cell Volume-Activated and Volume-Correlated Anion Channels in Mammalian Cells: Their Biophysical, Molecular, and Pharmacological Properties.

There are a number of mammalian anion channel types associated with cell volume changes. These channel types are classified into two groups: volume-activated anion channels (VAACs) and volume-correlated anion channels (VCACs). VAACs can be directly activated by cell swelling and include the volume-sensitive outwardly rectifying anion channel (VSOR), which is also called the volume-regulated anion channel; the maxi-anion channel (MAC or Maxi-Cl); and the voltage-gated anion channel, chloride channel (ClC)-2. VCACs can be facultatively implicated in, although not directly activated by, cell volume changes and include the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, the Ca2+-activated Cl- channel (CaCC), and the acid-sensitive (or acid-stimulated) outwardly rectifying anion channel. This article describes the phenotypical properties and activation mechanisms of both groups of anion channels, including accumulating pieces of information on the basis of recent molecular understanding. To that end, this review also highlights the molecular identities of both anion channel groups; in addition to the molecular identities of ClC-2 and CFTR, those of CaCC, VSOR, and Maxi-Cl were recently identified by applying genome-wide approaches. In the last section of this review, the most up-to-date information on the pharmacological properties of both anion channel groups, especially their half-maximal inhibitory concentrations (IC50 values) and voltage-dependent blocking, is summarized particularly from the standpoint of pharmacological distinctions among them. Future physiologic and pharmacological studies are definitely warranted for therapeutic targeting of dysfunction of VAACs and VCACs.

Construction and characterization of ribonuclease H2 knockout NIH3T3 cells.

Ribonuclease H (RNase H) specifically hydrolyzes the 5'-phosphodiester bonds of the RNA of RNA/DNA hybrid. Both types 1 and 2 RNases H act on the RNA strand of the hybrid, while only type 2 acts on the single ribonucleotide embedded in DNA duplex. In this study, to explore the role of mammalian type 2 RNase H (RNase H2) in cells, we constructed the RNase H2 knockout NIH3T3 cells (KO cells) by CRISPR/Cas9 system. KO cells hydrolyzed RNA strands in RNA/DNA hybrid, but not single ribonucleotides in DNA duplex, while wild-type NIH3T3 cells (WT cells) hydrolyzed both. Genomic DNA in the KO cells was more heavily hydrolyzed than in the WT cells by the alkaline or RNase H2 treatment, suggesting that the KO cells contained more ribonucleotides in genomic DNA than the WT cells. The growth rate of the KO cells was 60% of that of the WT cells. Expression of interferon-stimulated genes (ISGs) in the KO cells was not markedly elevated compared with the WT cells. These results suggest that in NIH3T3 cells, RNase H2 is crucial for suppressing the accumulation of ribonucleotides in genomic DNA but not for the expression of ISGs.

Preclinical Evaluation of AZ12601011 and AZ12799734, Inhibitors of Transforming Growth Factor β Superfamily Type 1 Receptors.

The transforming growth factor β (TGFβ) superfamily includes TGFβ, activins, inhibins, and bone morphogenetic proteins (BMPs). These extracellular ligands have essential roles in normal tissue homeostasis by coordinately regulating cell proliferation, differentiation, and migration. Aberrant signaling of superfamily members, however, is associated with fibrosis as well as tumorigenesis, cancer progression, metastasis, and drug-resistance mechanisms in a variety of cancer subtypes. Given their involvement in human disease, the identification of novel selective inhibitors of TGFβ superfamily receptors is an attractive therapeutic approach. Seven mammalian type 1 receptors have been identified that have context-specific roles depending on the ligand and the complex formation with the type 2 receptor. Here, we characterize the biologic effects of two transforming growth factor β receptor 1 (TGFBR1) kinase inhibitors designed to target TGFβ signaling. AZ12601011 [2-(2-pyridinyl)-4-(1H-pyrrolo[3,2-c]pyridin-1-yl)-6,7-dihydro-5H-cyclopenta[d]pyrimidine]; structure previously undisclosed] and AZ12799734 [4-({4-[(2,6-dimethyl-3-pyridinyl)oxy]-2-pyridinyl}amino)benzenesulfonamide] (IC50 = 18 and 47 nM, respectively) were more effective inhibitors of TGFβ-induced reporter activity than SB-431542 [4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide] (IC50 = 84 nM) and LY2157299 [4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide monohydrate]] (galunisertib) (IC50 = 380 nM). AZ12601011 inhibited phosphorylation of SMAD2 via the type 1 receptors activin A receptor type 1B (ALK4), TGFBR1, and activin A receptor type 1C (ALK7). AZ12799734, however, is a pan TGF/BMP inhibitor, inhibiting receptor-mediated phosphorylation of SMAD1 by activin A receptor type 1L, bone morphogenetic protein receptor type 1A, and bone morphogenetic protein receptor type 1B and phosphorylation of SMAD2 by ALK4, TGFBR1, and ALK7. AZ12601011 was highly effective at inhibiting basal and TGFβ-induced migration of HaCaT keratinocytes and, furthermore, inhibited tumor growth and metastasis to the lungs in a 4T1 syngeneic orthotopic mammary tumor model. These inhibitors provide new reagents for investigating in vitro and in vivo pathogenic processes and the contribution of TGFβ- and BMP-regulated signaling pathways to disease states.

Oxygen-Guided Radiation Therapy

It has been known for over 100years that tumor hypoxia, a near-universal characteristic of solid tumors, decreases the curative effectiveness of radiation therapy. However, to date, there are no reports that demonstrate an improvement in radiation effectiveness in a mammalian tumor on the basis of tumor hypoxia localization and local hypoxia treatment. For radiation targeting of hypoxic subregions in mouse fibrosarcoma, we used oxygen images obtained using pulse electron paramagnetic resonance pO2 imaging combined with 3D-printed radiation blocks. This achieved conformal radiation delivery to all hypoxic areas in FSa fibrosarcomas in mice. We demonstrate that treatment delivering a radiation boost to hypoxic volumes has a significant (P=.04) doubling of tumor control relative to boosts to well-oxygenated volumes. Additional dose to well-oxygenated tumor regions minimally increases tumor control beyond the 15% control dose to the entire tumor. If we can identify portions of the tumor that are more resistant to radiation, it might be possible to reduce the dose to more sensitive tumor volumes without significant compromise in tumor control. This work demonstrates in a single, intact mammalian tumor type that tumor hypoxia is a local tumor phenomenon whose treatment can be enhanced by local radiation. Despite enormous clinical effort to overcome hypoxic radiation resistance, to our knowledge this is the first such demonstration, even in preclinical models, of targeting additional radiation to hypoxic tumor to improve the therapeutic ratio.

Characteristics of Collagen-Rich Extracellular Matrix Hydrogels and Their Functionalization with Poly(ethylene glycol) Derivatives for Enhanced Biomedical Applications: A Review

The hydrogels of natural extracellular matrix (ECM) are excellent biomaterials with promising applications in the physiological manufacture of three-dimensional (3D) constructs that replicate native tissue-like architectures and function as cargo-delivery, 3D bioprinting, or injectable systems. ECM hydrogels retain the bioactivity to trigger key cellular processes in the tissue engineering and regenerative medicine (TERM) strategies. However, they lack suitable physicochemical properties, which restricts their applications in vivo. This demand that mechanical and degradation properties of the ECM hydrogels must be balanced against biological properties. By incorporating poly(ethylene glycol) (PEG) into mammalian type I collagen-rich ECM substrates, this task can be accomplished. This review is focused on the use of PEG derivatives, widely used in formulations of pharmaceutical products or in synthesis of biomedical polyurethanes, as a strategy to modulate both physical and biological properties of natural ECM hydrogels. The processing-property relationship in decellularized ECM hydrogels, as well as the main results when used in TERM, are discussed. A comparison of the characteristics of PEG-ECM hydrogels is provided in terms of the improvement of structure, mechanics, and degradation behavior. Finally, the benefits of producing PEG-ECM hydrogels according to in vitro and in vivo performance in different proofs-of-concept of emergent biomedical technologies are overviewed.

Function, Structure, and Transport Aspects of ZIP and ZnT Zinc Transporters in Immune Cells.

Zinc is an important trace metal in immune systems, and zinc transporters are involved in many immune responses. Recent advances have revealed the structural and biochemical bases for zinc transport across the cell membrane, with clinical implications for the regulation of zinc homeostasis in immune cells like dendritic cells, T cells, B cells, and mast cells. In this review, we discuss the function, structure, and transport aspects of two major mammalian zinc transporter types, importers and exporters. First, Zrt-/Irt-like proteins (ZIPs) mediate the zinc influx from the extracellular or luminal side into the cytoplasm. There are 14 ZIP family members in humans. They form a homo- or heterodimer with 8 transmembrane domains and extra-/intracellular domains of various lengths. Several ZIP members show specific extracellular domains composed of two subdomains, a helix-rich domain and proline-alanine-leucine (PAL) motif-containing domain. Second, ZnT (zinc transporter) was initially identified in early studies of zinc biology; it mediates zinc efflux as a counterpart of ZIPs in zinc homeostasis. Ten family members have been identified. They show a unique architecture characterized by a Y-shaped conformation and a large cytoplasmic domain. A precise, comprehensive understanding of the structures and transport mechanisms of ZIP and ZnT in combination with mice experiments would provide promising drug targets as well as a basis for identifying other transporters with therapeutic potential.

A targeted mass spectrometry method to screen collagen types I-V in the decellularized 3D extracellular matrix of the adult male rat thyroid

Here we have developed and validated an original LC-MS/MS SRM procedure flexible enough to quantitatively screen collagen types I-V in copies of the same type of stromal matrix prepared with different protocols of cell removal to retain the native 3D architecture of the ECM. In a first step, identification of tryptic sequences exclusive to specific chains (either α1 or α2) of mammalian collagen standards types I-V was pursued using a combination of LC-LIT-Orbitrap XL and LC-MS/MS SRM analyses. In a second step, the adult male rat thyroid was decellularized using three different protocols specifically set for engineering of bioartificial 3D thyroid organoids. In a third step, DNA analysis of the decellularized 3D thyroid stroma was pursued to exclude contamination by cell nuclear debris. In a final step, collagen standards and 3D thyroid matrices were digested using the same mechanical / enzymatic protocol, and quantitative profiles of collagen types I-V ensued using comparisons of ionic intensities between tryptic peptides of collagen standards and matrices, as derived from targeted LC-MS/MS SRM analysis. Collectively, the procedure allowed for detection and quantitation of collagen types I-V at a femtomolar level in thyroid gland stromal matrices initially maintaining their original 3D architecture, tryptically digested through a method common to collagen standards and thyroid ECM, with satisfactory reproducibility of results, moderate procedural cost, and limited analytical time.

Evaluation of the pathogenicity of mammalian orthoreovirus type 3 (MRV3) in germ-free gnotobiotic pigs and of the efficacy of an inactivated vaccine against MRV3 infection in neonatal conventional piglets

A novel U.S. strain of mammalian orthoreovirus type 3 (MRV3) isolated from diarrheic pigs in 2015 was reportedly highly pathogenic in pigs. In this study, we first developed an inactivated MRV3 vaccine and determined its protective efficacy against MRV3 infection in conventional neonatal piglets. A pathogenicity study was also conducted in gnotobiotic pigs to further assess the pathogenicity of MRV3. To evaluate if piglets could be protected against MRV3 infection after immunization of pregnant sows with an inactivated MRV3 vaccine, pregnant sows were vaccinated with 2 or 3 doses of the vaccine or with PBS buffer. Four-day-old piglets born to vaccinated and unvaccinated sows were subsequently challenged with MRV3. The results showed that piglets born from vaccinated sows had lower levels of fecal viral RNA shedding at 1, 3, and 4 days post-challenge, suggesting that the inactivated MRV3 vaccine can reduce MRV3 replication. Surprisingly, although the conventional piglets were infected, they did not develop severe enteric disease as reported previously. Therefore, in an effort to further definitively assess the pathogenicity of MRV3, we experimentally infected gnotobiotic pigs, a more sensitive model for pathogenicity study, with the wild-type MRV3 virus. The infected gnotobiotic piglets all survived and exhibited only very mild diarrhea in some pigs. Taken together, the results indicate that the novel strain of MRV3 recently isolated in the United States infected but caused only very mild diarrhea in pigs, and that maternal immunity acquired from sows vaccinated with an inactivated vaccine can reduce MRV3 replication in neonatal pigs.

Cryptochrome genes mediate photoperiodic responses in the cricket Modicogryllus siamensis

AbstractDiapause is a strategy used by many insects living in temperate zones to survive the winter. The most reliable environmental cue to induce diapause is photoperiod, for which day or night length is measured by a mechanism called the photoperiodic clock. Despite several studies in insects, the photoperiodic mechanisms remain unclear. In the present study, we show that cryptochromes (crys) mediate photoperiodic responses in the cricket Modicogryllus siamensis. Nymphal development of M. siamensis is highly dependent on photoperiod: under long‐day conditions, most nymphs become adults within 60 days after hatching by undergoing seven moults, whereas, under short‐day conditions, they take much longer (approximately 180 days), with an increased number of moults. Similar to most insects, the cricket has two cry genes: the Drosophila type cry (Ms'cry1) and the mammalian type cry (Ms'cry2). Ms'cry1 shows some constitutive expression, whereas Ms'cry2 is rhythmically expressed under both long‐day and short‐day conditions. Parental RNA interference against either or both of the two cry genes partially prevents the long‐day responses, whereas inhibition of Ms'cry1 or Ms'cry1/Ms'cry2 enhances the short‐day response with a further delay of adult emergence or its complete prevention, respectively. Nymphal RNA interference of Ms'crys at the fourth‐instar stage delays adult emergence and increases the number of moults under long‐day conditions. We hypothesize that Ms'cry genes are involved not only in the photoperiodic mechanism, but also in the regulatory mechanism of nymphal development downstream of the photoperiodic clock in the cricket M. siamensis.

Characterization of a replicating expanded tropism oncolytic reovirus carrying the adenovirus E4orf4 gene.

While the mammalian orthoreovirus type 3 dearing (reovirus T3D) infects many different tumour cells, various cell lines resist the induction of reovirus-mediated cell death. In an effort to increase the oncolytic potency, we introduced transgenes into the S1 segment of reovirus T3D. The adenovirus E4orf4 gene was selected as transgene since the encoded E4orf4 protein induces cell death in transformed cells. The induction of cell death by E4orf4 depends in part on its binding to phosphatase 2A (PP2A). In addition to the S1-E4orf4 reovirus, two other reoviruses were employed in our studies. The reovirus rS1-RFA encodes an E4orf4 double-mutant protein that cannot interact with PP2A and the rS1-iLOV virus encoding the fluorescent marker iLOV as a reporter. The replacement of the codons for the junction adhesion molecule-A (JAM-A) binding head domain of the truncated spike protein blocks the entry of these recombinant viruses via the reovirus receptor JAM-A. Instead these viruses rely on internalization via binding to sialic acids on the cell surface. This expands their tropism and allows infection of JAM-A-deficient tumour cells. Here we not only demonstrate the feasibility of this approach but also established that the cytolytic activity of these recombinant viruses is largely transgene independent.

Preliminary study of guard hair morphology in four dog breeds.

Morphological differences between mammalian hair types are useful for species distinction. Differences in animal hair structure are observed between species, whereas there may be breed differences within a species. Guard hair structure may vary between different dog breeds. Hair samples were collected from 73 dogs of four breed types (wire-haired Dachshund, Labrador retriever, German shepherd dog and Yorkshire terrier). Hair was measured and analysed by scanning electron microscopy. The following were evaluated: hair thickness; medulla to hair cross-sectional area ratio; scale density, pattern and margins; and medulla shape, composition and structure. There were significant differences in all measured parameters between breeds. These study findings may be useful for the study of classifying dog breeds and for forensic identification of a dog breed through hair sampling.

The role of type I interferons (IFNs) in the regulation of chicken macrophage inflammatory response to bacterial challenge

Mammalian type I interferons (IFNα/β) are known to modulate inflammatory processes in addition to their antiviral properties. Indeed, virus-induced type I interferons regulate the mammalian phagocyte immune response to bacteria during superinfections. However, it remains unresolved whether type I IFNs similarly impact the chicken macrophage immune response. We first evidenced that IFNα and IFNβ act differently in terms of gene expression stimulation and activation of intracellular signaling pathways in chicken macrophages. Next, we showed that priming of chicken macrophages with IFNα increased bacteria uptake, boosted bacterial-induced ROS/NO production and led to an increased transcriptional expression or production of NOS2/NO, IL1B/IL-1β and notably IFNB/IFNβ. Neutralization of IFNβ during bacterial challenge limited IFNα-induced augmentation of the pro-inflammatory response. In conclusion, we demonstrated that type I IFNs differently regulate chicken macrophage functions and drive a pro-inflammatory response to bacterial challenge. These findings shed light on the diverse functions of type I IFNs in chicken macrophages.

A versatile plasmid system for reconstitution and analysis of mammalian ubiquitination cascades in yeast.

Ubiquitination is a posttranslational protein modification that regulates most aspects of cellular life. The sheer number of ubiquitination enzymes that are present in a mammalian cell, over 700 in total, has thus far hampered the analysis of distinct protein ubiquitination cascades in a cellular context. To overcome this complexity we have developed a versatile vector system that allows the reconstitution of specific ubiquitination cascades in the model eukaryote Saccharomyces cerevisae (baker’s yeast). The vector system consists of 32 modular yeast shuttle plasmids allowing inducible or constitutive expression of up to four proteins of interest in a single cell. To demonstrate the validity of the system, we show that co-expression in yeast of the mammalian HECT type E3 ubiquitin ligase E6AP (E6-Associated Protein) and a model substrate faithfully recapitulates E6AP-dependent substrate ubiquitination and degradation. In addition, we show that the endogenous sumoylation pathway of S. cerevisiae can specifically sumoylate mouse PML (Promyelocytic leukemia protein). In conclusion, the yeast vector system described in this paper provides a versatile tool to study complex post-translational modifications in a cellular setting.

An experimental toolbox for characterization of mammalian collagen type I in biological specimens.

Collagen type I is the most abundant extracellular matrix protein, and collagen type I supramolecular assemblies (e.g., tissue grafts, biomaterials and cell-assembled systems) are used extensively in tissue engineering and regenerative medicine. Many studies, for convenience or economic reasons, do not accurately determine collagen type I purity, concentration, solubility and extent of cross-linking in biological specimens, frequently resulting in erroneous conclusions. In this protocol, we describe solubility; normal, reduced and delayed (interrupted) SDS-PAGE; hydroxyproline; Sircol collagen and Pierce BCA protein; denaturation temperature; ninhydrin/trinitrobenzene sulfonic acid; and collagenase assays and assess them in a diverse range of biological samples (e.g., tissue samples; purified solutions or lyophilized materials; 3D scaffolds, such as sponges and hydrogels; and cell media and layers). Collectively, the described protocols provide a comprehensive, yet fast and readily implemented, toolbox for collagen type I characterization in any biological specimen.

Anatomical and Physiological Similarities of Kidney in Different Experimental Animals Used for Basic Studies

Introduction: Before the experiment on kidney disorder, it is important to know the basic physiologies of the animal kidney because the anatomical and physiological features of the kidney are varying in different kind of experimental animals used for the basic study. This study focus on the variations in the nephron, a morphological process of podocytes, a path of the renal arteries, a position of the medulla and renal cortex are unusual in different animals. In human, middle and caudal lobe is larger while in rat small rounded cranial lobe present in comparison to other experimental animals. Statistically the mean length 10.40 ± 0.90 cm, width 4.20 ± 0.70 cm, thickness 1.5 ± 0.20 cm, and weight 136.71 ± 31.74 g; while in rat the mean length 1.54 ± 0.02 cm, width 0.85 ± 0.07 cm, thickness 0.73 ± 0.02 cm and weight 0.64 ± 0.03 g. Human has shorter loops of Henle, which dip only into the outer medulla while in monkey both the renal cortex and medulla are higher in numbers than in dogs. The renal lobule of mallard species was comparatively divided into two equal cortical and medullary regions while in chickens the cortical region was the larger as compared to medullary region resembling numerous mammalian types and few of swine type. In contrast, the severity of tubular degeneration, inflammation, fibrosis, tubular epithelial single-cell necrosis, and decreased normal parenchyma is responsible for proteinuria. The morphological process of podocyte development has been reported in the classic kidney of human, horse, goat, monkey, rabbit, cat, dog, chicken, medaka fish, rat and guinea pig indicating that it would be a good model system for studying renal regeneration.

Existence of nicotinic receptors in a subset of type I vestibular hair cells of guinea pigs

In mammals, vestibular hair cells (VHCs) are classified as type I and II according to morphological criteria. Acetylcholine (ACh) is identified as the primary efferent neurotransmitter. To date, cholinergic activities have been reported in mammalian type II VHCs, but similar activities in type I VHCs have not been pursued presumably because the body of type I VHCs were suggested to be totally surrounded by afferent nerve calyces. A few reports showed that part of type I VHCs were incompletely surrounded by calyces and received contact from the efferent nerve endings in the mammals studied. The possibility of the expression of cholinergic receptors, their subunit composition, and their function in mammals’ type I VHCs are still unclear. In this study, nicotinic responses were investigated by the whole-cell patch clamp technique in isolated type I VHCs of guinea pigs. Of the cells, 7.3% were sensitive to cholinergic agonists and showed an excitatory current at −40 mV which was not sensitive to nifedipine, iberiotoxin (IBTX), and apamin. The main carriers of this current were Na+ and K+. The rank order of activation potency was nicotine > 1,1-dimethyl-4-phenyl-piperazinium (DMPP) > ACh. These nicotinic ACh receptors (nAChRs) were not blocked by strychnine and α-bungarotoxin (α-BTX), but sensitive to d-tubocurarine (dTC) and mecamylamine (Mec). The findings provide physiological evidence that some subtypes of nAChRs may be located in a subset of type I VHCs, which were different from α9α10 nAChRs.

Baculovirus-assisted Reovirus Infection in Monolayer and Spheroid Cultures of Glioma cells

The mammalian orthoreovirus Type 3 Dearing has great potential as oncolytic agent in cancer therapy. One of the bottlenecks that hampers its antitumour efficacy in vivo is the limited tumour-cell infection and intratumoural distribution. This necessitates strategies to improve tumour penetration. In this study we employ the baculovirus Autographa californica multiple nucleopolyhedrovirus as a tool to expand the reovirus’ tropism and to improve its spread in three-dimensional tumour-cell spheroids. We generated a recombinant baculovirus expressing the cellular receptor for reovirus, the Junction Adhesion Molecule-A, on its envelope. Combining these Junction Adhesion Molecule-A-expressing baculoviruses with reovirus particles leads to the formation of biviral complexes. Exposure of the reovirus-resistant glioblastoma cell line U-118 MG to the baculovirus-reovirus complexes results in efficient reovirus infection, high reovirus yields, and significant reovirus-induced cytopathic effects. As compared to the reovirus-only incubations, the biviral complexes demonstrated improved penetration and increased cell killing of three-dimensional U-118 MG tumour spheroids. Our data demonstrate that reovirus can be delivered with increased efficiency into two- and three-dimensional tumour-cell cultures via coupling the reovirus particles to baculovirus. The identification of baculovirus’ capacity to penetrate into tumour tissue opens novel opportunities to improve cancer therapy by improved delivery of oncolytic viruses into tumours.