Anatomy and Physiology Faculty Research and Publications

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Open Access
Computer Assisted Learning: Assessment of the Veterinary Virtual Anatomy Education Software IVALA™
(2018-06-19) Little, William Brady; Artemiou, Elpida; Conan, Anne; Sparks, Cathryn
Although cadaveric dissection has historically been the cornerstone of anatomical education, it comes at the cost of some emotional, moral, safety, and environmental concerns. Computer assisted learning (CAL) programs are an increasingly common solution to these issues; however, research regarding the efficacy of high fidelity simulation is limited. The traditional first semester veterinary gross anatomy course curriculum at Ross University School of Veterinary Medicine (RUSVM) was supplemented with a web based virtual anatomy program, IVALA™ (www.ivalalearn.com). The purpose of this study was to assess the relationship between supplementary use of the IVALA™ program and student examination scores, and to measure student perception surrounding IVALA™. IVALA™ uses an interactive virtual canine specimen that enables students to identify, move, rotate, magnify, and remove individual anatomic structures while providing a text description of each selected anatomic point. Fifty-six first semester RUSVM students who supplemented their anatomic learning with the IVALA™ program performed significantly higher on examinations compared to students (n = 123) that did not (p = 0.003). Students’ overall perception toward IVALA™ was enjoyable (mean = 3.8 out of a 5-point Likert scale) and beneficial to their knowledge of anatomy (mean = 3.7); however, students did not support replacing cadaveric dissection with CAL (mean = 2.1). CAL can effectively supplement learning outcomes for veterinary anatomy.
Open Access
Comparative Functional Dynamics Studies on the Enzyme Nano-bio Interface
(2018-08-08) Thomas, Spencer E.; Comer, Jeffrey; Kim, Min Jung; Marroquin, Shanna; Murthy, Vaibhav; Ramani, Meghana; Hopke, Tabetha Gaile; McCall, Jayden; Choi, Seong-O; DeLong, Robert K.
Comparative functional dynamics studies on the enzyme nano-bio interface Spencer E Thomas,1,2,* Jeffrey Comer,1,* Min Jung Kim,1 Shanna Marroquin,1 Vaibhav Murthy,1 Meghana Ramani,1 Tabetha Gaile Hopke,2 Jayden McCall,1 Seong-O Choi,1 Robert K DeLong1 1Nanotechnology Innovation Center of Kansas State, Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA; 2Department of Biomedical Science, Missouri State University, Springfield, MO, USA *These authors contributed equally to this work Introduction: Biomedical applications of nanoparticles (NPs) as enzyme inhibitors have recently come to light. Oxides of metals native to the physiological environment (eg, Fe, Zn, Mg, etc.) are of particular interest—especially the functional consequences of their enzyme interaction. Materials and methods: Here, Fe2O3, zinc oxide (ZnO), magnesium oxide (MgO) and nickel oxide (NiO) NPs are compared to copper (Cu) and boron carbide (B4C) NPs. The functional impact of NP interaction to the model enzyme luciferase is determined by 2-dimensional fluorescence difference spectroscopy (2-D FDS) and 2-dimensional photoluminescence difference spectroscopy (2-D PLDS). By 2-D FDS analysis, the change in maximal intensity and in 2-D FDS area under the curve (AUC) is in the order Cu~B4C>ZnO>NiO>>Fe2O3>MgO. The induced changes in protein conformation are confirmed by tryptic digests and gel electrophoresis. Results: Analysis of possible trypsin cleavage sites suggest that cleavage mostly occurs in the range of residues 112–155 and 372–439, giving a major 45 kDa band. By 2-D PLDS, it is found that B4C NPs completely ablate bioluminescence, while Cu and Fe2O3 NPs yield a unique bimodal negative decay rate, -7.67×103 and -3.50×101 relative light units respectively. Cu NPs, in particular, give a remarkable 271% change in enzyme activity. Molecular dynamics simulations in water predicted that the surfaces of metal oxide NPs become capped with metal hydroxide groups under physiological conditions, while the surface of B4C becomes populated with boronic acid or borinic acid groups. These predictions are supported by the experimentally determined zeta potential. Thin layer chromatography patterns further support this conception of the NP surfaces, where stabilizing interactions were in the order ionic>polar>non-polar for the series tested. Conclusion: Overall the results suggest that B4C and Cu NP functional dynamics on enzyme biochemistry are unique and should be examined further for potential ramifications on other model, physiological or disease-relevant enzymes. Keywords: 2-dimensional fluorescence difference, 2-D FDS, AUC, corrected light intensity emitted, emission wavelength, excitation wavelength, wavelength of max intensity, RLU
Open Access
Claudin expression during early postnatal development of the murine cochlea
(2018-01-25) Kudo, Takayuki; Wangemann, Philine; Marcus, Daniel C.; wange; marcus
Background: Claudins are major components of tight junctions, which form the paracellular barrier between the cochlear luminal and abluminal fluid compartments that supports the large transepithelial voltage difference and the large concentration differences of K+, Na+ and Ca2+ needed for normal cochlear function. Claudins are a family of more than 20 subtypes, but our knowledge about expression and localization of each subtype in the cochlea is limited. Results: We examined by quantitative RT-PCR the expression of the mRNA of 24 claudin isoforms in mouse cochlea during postnatal development and localized the expression in separated fractions of the cochlea. Transcripts of 21 claudin isoforms were detected at all ages, while 3 isoforms (Cldn-16, ??17 and ??18) were not detected. Claudins that increased expression during development include Cldn-9, ??13, ??14, ??15, and -19v2, while Cldn-6 decreased. Those that do not change expression level during postnatal development include Cldn-1, ??2, ??3, ??4, ??5, ??7, ??8, ?10v1, ?10v2, ??11, ??12, ?19v1, ??20, ??22, and???23. Our investigation revealed unique localization of some claudins. In particular, Cldn-13 expression rapidly increases during early development and is mainly expressed in bone but only minimally in the lateral wall (including stria vascularis) and in the medial region (including the organ of Corti). No statistically significant changes in expression of Cldn-11, ??13, or ??14 were found in the cochlea of Slc26a4 ?/? mice compared to Slc26a4 +/? mice. Conclusions: We demonstrated developmental patterns of claudin isoform transcript expression in the murine cochlea. Most of the claudins were associated with stria vascularis and organ of Corti, tissue fractions rich in tight junctions. However, this study suggests a novel function of Cldn-13 in the cochlea, which may be linked to cochlear bone marrow maturation.
Open Access
Dual-nozzle spray deposition process for improving the stability of proteins in polymer microneedles
(2017-10-04) Kim, Min Jung; Parka, Seok Chan; Choi, Seong-O; mj1217; schpark; sochoi; Kim, Min Jung; Parka, Seok Chan; Choi, Seong-O
Polymer microneedles are an attractive way of transdermal delivery of various pharmaceutical compounds. Fabrication of drug-encapsulating polymer microneedles, however, often involves processing conditions unfavorable for maintaining the stability of drugs, including highly concentrated formulations, high temperature and long drying time. The stability of labile substances in biodegradable polymer matrices could also be significantly reduced by the use of organic solvents and emulsification. In this paper, we reported a new fabrication technique called the dual-nozzle spray deposition process, which utilizes a separate deposition of drug and polymer solutions, as a potential way to address the stability issue associated with current microneedle fabrication processes. A model protein, bovine serum albumin (BSA), was successfully incorporated into both water-soluble poly(vinyl alcohol) (PVA) and biodegradable poly(lactic-co-glycolic acid) (PLGA) microneedles by the dual-nozzle spray deposition process. The conformational change of BSA examined by circular dichroism and fluorescence spectroscopy suggests that the developed process helps maintain the structural stability of BSA during encapsulation in both PVA and PLGA matrices. We anticipate that the dual-nozzle spray deposition process would improve the stability of drugs by reducing adverse interaction with solvents and eliminating the emulsification process. Also, the developed process could be an attractive approach to fabricating polymer-based drug delivery devices.
Open Access
Estimation of tulathromycin depletion in plasma and milk after subcutaneous injection in lactating goats using a nonlinear mixed-effects pharmacokinetic modeling approach
(2016-11-18) Lin, Zhoumeng; Cuneo, M.; Rowe, J. D.; Li, M. J.; Tell, L. A.; Allison, S.; Carlson, J.; Riviere, Jim E.; Gehring, Ronette; zhoumeng; jriviere; rgehring; Lin, Zhoumeng; Riviere, Jim E.; Gehring, Ronette
Background: Extra-label use of tulathromycin in lactating goats is common and may cause violative residues in milk. The objective of this study was to develop a nonlinear mixed-effects pharmacokinetic (NLME-PK) model to estimate tulathromycin depletion in plasma and milk of lactating goats. Eight lactating goats received two subcutaneous injections of 2.5 mg/kg tulathromycin 7 days apart; blood and milk samples were analyzed for concentrations of tulathromycin and the common fragment of tulathromycin (i.e., the marker residue CP-60,300), respectively, using liquid chromatography mass spectrometry. Based on these new data and related literature data, a NLME-PK compartmental model with first-order absorption and elimination was used to model plasma concentrations and cumulative excreted amount in milk. Monte Carlo simulations with 100 replicates were performed to predict the time when the upper limit of the 95% confidence interval of milk concentrations was below the tolerance. Results: All animals were healthy throughout the study with normal appetite and milk production levels, and with mild-moderate injection-site reactions that diminished by the end of the study. The measured data showed that milk concentrations of the marker residue of tulathromycin were below the limit of detection (LOD = 1.8 ng/ml) 39 days after the second injection. A 2-compartment model with milk as an excretory compartment best described tulathromycin plasma and CP-60,300 milk pharmacokinetic data. The model-predicted data correlated with the measured data very well. The NLME-PK model estimated that tulathromycin plasma concentrations were below LOD (1.2 ng/ml) 43 days after a single injection, and 62 days after the second injection with a 95% confidence. These estimated times are much longer than the current meat withdrawal time recommendation of 18 days for tulathromycin in non-lactating cattle. Conclusions: The results suggest that twice subcutaneous injections of 2.5 mg/kg tulathromycin are a clinically safe extra-label alternative approach for treating pulmonary infections in lactating goats, but a prolonged withdrawal time of at least 39 days after the second injection should be considered to prevent violative residues in milk and any dairy goat being used for meat should have an extended meat withdrawal time.
Open Access
Complete genome sequence of a sub-subgenotype 2.1i isolate of classical swine fever virus from China
(2017-04-06) Zhang, B.; Mi, S.; Bao, F.; Guo, H.; Tu, C.; Shi, Jishu; Gong, W.; jshi; Shi, Jishu
The complete genome sequence of a sub-subgenotype 2.1i isolate of classical swine fever virus (CSFV), GD317/2011, was determined. Notably, GD317/2011 is distant from the sub-subgenotype 2.1b isolate HEBZ at genes of Erns, E1, E2, P7, NS2, NS5A and the 3=-nontranslated region (3=-NTR) but is closely related to that at genes of Npro, Core, NS3, NS4A, NS4B, and NS5B. © 2017 Zhang et al.
Open Access
Serum Metabolomic Profiling of Piglets Infected with Virulent Classical Swine Fever Virus
(2017-04-27) Gong, Wenjie J.; Jia, J. J.; Zhang, B. K.; Mi, S. J.; Zhang, L.; Xie, X. M.; Guo, H. C.; Shi, Jishu; Tu, C. C.; jshi; Shi, Jishu
Classical swine fever (CSF) is a highly contagious swine infectious disease and causes significant economic losses for the pig industry worldwide. The objective of this study was to determine whether small molecule metabolites contribute to the pathogenesis of CSF. Birefly, serum metabolomics of CSFV Shimen strain-infected piglets were analyzed by ultraperformance liquid chromatography/electrospray ionization time-of-flight mass spectrometry (UPLC/ESI-Q-TOF/MS) in combination with multivariate statistical analysis. In CSFV-infected piglets at days 3 and 7 post-infection changes were found in metabolites associated with several key metabolic pathways, including tryptophan catabolism and the kynurenine pathway, phenylalanine metabolism, fatty acid and lipid metabolism, the tricarboxylic acid and urea cycles, branched-chain amino acid metabolism, and nucleotide metabolism. Several pathways involved in energy metabolism including fatty acid biosynthesis and beta-oxidation, branched-chain amino acid metabolism, and the tricarboxylic acid cycle were significantly inhibited. Changes were also observed in several metabolites exclusively associated with gut microbiota. The metabolomic profiles indicate that CSFV-host gut microbiome interactions play a role in the development of CSF.
Open Access
Limitations of MIC as sole metric of pharmacodynamic response across the range of antimicrobial susceptibilities within a single bacterial species
(2016-12-01) Wen, X. S.; Gehring, Ronette; Stallbaumer, A.; Riviere, Jim E.; Volkova, Victoriya V.; rgehring; jriviere; vv88; Gehring, Ronette; Riviere, Jim E.; Volkova, Victoriya V.
The minimum inhibitory concentration (MIC) of an antimicrobial drug for a bacterial pathogen is used as a measure of the bacterial susceptibility to the drug. However, relationships between the antimicrobial concentration, bacterial susceptibility, and the pharmacodynamic (PD) inhibitory effect on the bacterial population are more complex. The relationships can be captured by multi-parameter models such as the E-max model. In this study, time-kill experiments were conducted with a zoonotic pathogen Pasteurella multocida and the fluoroquinolone enrofloxacin. Pasteurella multocida isolates with enrofloxacin MIC of 0.01 mu g/mL, 1.5 mu g/mL, and 2.0 mu g/mL were used. An additive inhibitory E-max model was fitted to the data on bacterial population growth inhibition at different enrofloxacin concentrations. The values of PD parameters such as maximal growth inhibition, concentration achieving a half of the maximal inhibition, and Hill coefficient that captures steepness of the relationships between the concentration and effect, varied between the isolate with low MIC and less susceptible isolates. While enrofloxacin PD against the isolate with low MIC exhibited the expected concentration-dependent characteristics, the PD against the less susceptible isolates demonstrated time-dependent characteristics. The results demonstrate that bacterial antimicrobial susceptibility may need to be described by a combination of parameters rather than a single parameter of the MIC.
Open Access
Exopolysaccharides extracted from Parachlorella kessleri inhibit colon carcinoma growth in mice via stimulation of host antitumor immune responses
(2017-04-05) Ishiguro, S.; Uppalapati, D.; Goldsmith, Z.; Robertson, D.; Hodge, J.; Holt, H.; Nakashima, A.; Turner, K.; Tamura, Masaaki; masaakit; Tamura, Masaaki
The newly purified extracellular polysaccharides (exopolysaccharides) from Parachlorella kessleri (PCEPS) were evaluated on their antitumor and immunomodulatory effects in cell culture and mouse colon carcinoma peritoneal dissemination model. In two-dimensional cell culture, the PCEPS treatment inhibited cell growth of both murine and human colon carcinoma cells in a dose- and time-dependent manner. In contrast, the growth of mouse splenocytes (SPLs) and bone marrow cells (BMCs) were stimulated by the treatment with PCEPS. The treatment with PCEPS also increased specific subpopulations of the cells in BMCs: antigen presenting cells (CD19(+) B cells, 33D1(+) dendritic cells and CD68(+) macrophage) and CD8(+) cytotoxic T cells. In three-dimensional spheroid culture, spheroid growth of CT26 cells co-cultured with HL-60 human neutrophilic promyeloblasts and Jurkat cells (human lymphoblasts), but not THP1 human monocyte/macrophage was significantly attenuated by PCEPS treatment. In a mouse CT26 colon carcinoma peritoneal dissemination model, intraperitoneal injection of PCEPS (10 mg/kg, twice per week) significantly attenuated the growth of CT26 colon carcinoma in syngeneic mice. The present study suggests that PCEPS inhibits colon carcinoma growth via direct cell growth inhibition and a stimulation of the host antitumor immune responses. Taken together, the current study suggests that exopolysaccharides derived from Parachlorella kessleri contain significant bioactive materials that inhibit colon carcinoma growth.
Open Access
Exercise-induced pulmonary hemorrhage: where are we now?
(2016-11-07) Poole, David C.; Erickson, Howard H.; dcpoole; hhe; Poole, David C.; Erickson, Howard H.
As the Thoroughbreds race for the final stretch, 44 hooves flash and thunder creating a cacophony of tortured air and turf. Orchestrated by selective breeding for physiology and biomechanics, expressed as speed, the millennia-old symphony of man and beast reaches its climax. At nearly 73 kilometers per hour (45 mph) over half a ton of flesh and bone dwarfs its limpet-like jockey as, eyes wild and nostrils flaring, their necks stretch for glory. Beneath each resplendent livery-adorned, latherin-splattered coat hides a monstrous heart trilling at 4 beats per second, and each minute, driving over 400 L (105 gallons) of oxygen-rich blood from lungs to muscles. Matching breath to stride frequency, those lungs will inhale 16 L (4 gallons) of air each stride moving >1,000 L/min in and out of each nostril - and yet failing. Engorged with blood and stretched to breaking point, those lungs can no longer redden the arterial blood but leave it dusky and cyanotic. Their exquisitely thin blood-gas barrier, a mere 10.5 mu m thick (1/50,000 of an inch), ruptures, and red cells invade the lungs. After the race is won and lost, long after the frenetic crowd has quieted and gone, that blood will clog and inflame the airways. For a few horses, those who bleed extensively, it will overflow their lungs and spray from their nostrils incarnadining the walls of their stall: a horrifically poignant canvas that strikes at horse racing's very core. That exercise-induced pulmonary hemorrhage (EIPH) occurs is a medical and physiological reality. That every reasonable exigency is not taken to reduce/prevent it would be a travesty. This review is not intended to provide an exhaustive coverage of EIPH for which the reader is referred to recent reviews, rather, after a brief reminder of its physiologic and pathologic bases, focus is brought on the latest developments in EIPH discovery as this informs state-of-the-art knowledge, the implementation of that knowledge and recommendations for future research and treatment.
Open Access
Overexpression of Parkinson's Disease-Associated Mutation LRRK2 G2019S in Mouse Forebrain Induces Behavioral Deficits and alpha-Synuclein Pathology
(2017-03-06) Xiong, Y. L.; Neifert, S.; Karuppagounder, S. S.; Stankowski, J. N.; Lee, B. D.; Grima, J. C.; Chen, G. X.; Ko, H. S.; Lee, Y.; Swing, D.; Tessarollo, L.; Dawson, T. M.; Dawson, V. L.; yulanx; Xiong, Yulan
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified as an unambiguous cause of late-onset, autosomal-dominant familial Parkinson's disease (PD) and LRRK2 mutations are the strongest genetic risk factor for sporadic PD known to date. A number of transgenic mice expressing wild-type or mutant LRRK2 have been described with varying degrees of LRRK2-related abnormalities and modest pathologies. None of these studies directly addressed the role of the kinase domain in the changes observed and none of the mice present with robust features of the human disease. In an attempt to address these issues, we created a conditional LRRK2 G2019S (LRRK2 GS) mutant and a functionally negative control, LRRK2 G2019S/D1994A (LRRK2 GS/DA). Expression of LRRK2 GS or LRRK2 GS/DA was conditionally controlled using the tet-off system in which the presence of tetracycline-transactivator protein (tTA) with a CAMKII alpha promoter (CAMKII alpha-tTA) induced expression of TetP-LRRK2 GS or TetP-LRRK2 GS/DA in the mouse forebrain. Overexpression of LRRK2 GS in mouse forebrain induced behavioral deficits and alpha-synuclein pathology in a kinase-dependent manner. Similar to other genetically engineered LRRK2 GS mice, there was no significant loss of dopaminergic neurons. These mice provide an important new tool to study neurobiological changes associated with the increased kinase activity from the LRRK2 G2019S mutation, which may ultimately lead to a better understanding of not only the physiologic actions of LRRK2, but also potential pathologic actions that underlie LRRK2 GS-associated PD.
Open Access
Determinants of Alanine Dipeptide Conformational Equilibria on Graphene and Hydroxylated Derivatives
(2017-03-14) Poblete, Horacio; Miranda-Carvajal, Ingrid; Comer, Jeffrey R.; jeffcomer; Poblete, Horacio; Comer, Jeffrey R.
Understanding the interaction of carbon nanomaterials with proteins is essential for determining the potential effects of these materials on health and in the design of biotechnology based on them. Here we leverage explicit-solvent molecular simulation and multidimensional free-energy calculations to investigate how adsorption to carbon nanomaterial surfaces affects the conformational equilibrium of alanine dipeptide, a widely used model of protein backbone structure. We find that the two most favorable structures of alanine dipeptide on graphene (or large carbon nanotubes) correspond to the two amide linkages lying in the same plane, flat against the surface, rather than the nonplanar ?-helix-like and ?-sheet-like conformations that predominate in aqueous solution. On graphenic surfaces, the latter conformations are metastable and most often correspond to amide?? stacking of the N-terminal amide. The calculations highlight the key role of amide?? interactions in determining the conformational equilibrium. Lesser but significant contributions from hydrogen bonding to the high density interfacial water layer or to the hydroxy groups of hydroxylated graphene also define the most favorable conformations. This work should yield insight on the influence of carbon nanotubes, graphene, and their functionalized derivatives on protein structure.
Open Access
Subdiffusion in Membrane Permeation of Small Molecules
(2016-11-02) Chipot, Christophe; Comer, Jeffrey R.; jeffcomer; Comer, Jeffrey R.
Within the solubility–diffusion model of passive membrane permeation of small molecules, translocation of the permeant across the biological membrane is traditionally assumed to obey the Smoluchowski diffusion equation, which is germane for classical diffusion on an inhomogeneous free-energy and diffusivity landscape. This equation, however, cannot accommodate subdiffusive regimes, which have long been recognized in lipid bilayer dynamics, notably in the lateral diffusion of individual lipids. Through extensive biased and unbiased molecular dynamics simulations, we show that one-dimensional translocation of methanol across a pure lipid membrane remains subdiffusive on timescales approaching typical permeation times. Analysis of permeant motion within the lipid bilayer reveals that, in the absence of a net force, the mean squared displacement depends on time as t0.7, in stark contrast with the conventional model, which assumes a strictly linear dependence. We further show that an alternate model using a fractional-derivative generalization of the Smoluchowski equation provides a rigorous framework for describing the motion of the permeant molecule on the pico- to nanosecond timescale. The observed subdiffusive behavior appears to emerge from a crossover between small-scale rattling of the permeant around its present position in the membrane and larger-scale displacements precipitated by the formation of transient voids.
Open Access
Predicting Adsorption Affinities of Small Molecules on Carbon Nanotubes Using Molecular Dynamics Simulation
(2015-10-27) Comer, Jeffrey R.; Chen, Ran; Poblete, Horacio; Vergara-Jaque, Ariela; Riviere, Jim E.; jeffcomer; ranchen; jriviere; Comer, Jeffrey R.; Chen, Ran; Poblete, Horacio; Vergara-Jaque, Ariela; Riviere, Jim E.
Computational techniques have the potential to accelerate the design and optimization of nanomaterials for applications such as drug delivery and contaminant removal; however, the success of such techniques requires reliable models of nanomaterial surfaces as well as accurate descriptions of their interactions with relevant solutes. In the present work, we evaluate the ability of selected models of naked and hydroxylated carbon nanotubes to predict adsorption equilibrium constants for about 30 small aromatic compounds with a variety of functional groups. The equilibrium constants determined using molecular dynamics coupled with free-energy calculation techniques are directly compared to those derived from experimental measurements. The calculations are highly predictive of the relative adsorption affinities of the compounds, with excellent correlation (r ? 0.9) between calculated and measured values of the logarithm of the adsorption equilibrium constant. Moreover, the agreement in absolute terms is also reasonable, with average errors of less than one decade. We also explore possible effects of surface loading, although we demonstrate that they are negligible for the experimental conditions considered. Given the degree of reliability demonstrated, we move on to employing the in silico techniques in the design of nanomaterials, using the optimization of adsorption affinity for the herbacide atrazine as an example. Our simulations suggest that, compared to other modifications of graphenic carbon, polyvinylpyrrolidone conjugation gives the highest affinity for atrazine—substantially greater than that of graphenic carbon alone—and may be useful as a nanomaterial for delivery or sequestration of atrazine.
Open Access
Epithelial cell stretching and luminal acidification lead to a retarded development of stria vascularis and deafness in mice lacking pendrin
(2011-03-14) Kim, H. M.; Wangemann, Antje Philine; wange; Wangemann, Philine; Kim, H. M.
Loss-of-function mutations of SLC26A4/pendrin are among the most prevalent causes of deafness. Deafness and vestibular dysfunction in the corresponding mouse model, Slc26a4-/-, are associated with an enlargement and acidification of the membranous labyrinth. Here we relate the onset of expression of the HCO3- transporter pendrin to the luminal pH and to enlargement-associated epithelial cell stretching. We determined expression with immunocytochemistry, cell stretching by digital morphometry and pH with double-barreled ion-selective electrodes. Pendrin was first expressed in the endolymphatic sac at embryonic day (E) 11.5, in the cochlear hook-region at E13.5, in the utricle and saccule at E14.5, in ampullae at E16.5, and in the upper turn of the cochlea at E17.5. Epithelial cell stretching in Slc26a4-/- mice began at E14.5. pH changes occurred first in the cochlea at E15.5 and in the endolymphatic sac at E17.5. At postnatal day 2, stria vascularis, outer sulcus and Reissner's membrane epithelial cells, and utricular and saccular transitional cells were stretched, whereas sensory cells in the cochlea, utricle and saccule did not differ between Slc26a4+/- and Slc26a4-/- mice. Structural development of stria vascularis, including vascularization, was retarded in Slc26a4-/- mice. In conclusion, the data demonstrate that the enlargement and stretching of non-sensory epithelial cells precedes luminal acidification in the cochlea and the endolymphatic sac. Stretching and luminal acidification may alter cell-to-cell communication and lead to the observed retarded development of stria vascularis, which may be an important step on the path to deafness in Slc26a4-/- mice, and possibly in humans, lacking functional pendrin expression.
Open Access
Two-Dimensional Fluorescence Difference Spectroscopy to Characterize Nanoparticles and their Interactions
Hurst, Miranda N.; DeLong, Robert K.; robertdelong; DeLong, Robert K.; Hurst, Miranda N.
Two dimensional fluorescence difference spectroscopy (2D FDS) detects nanoparticle interactions following surface functionalization and biomolecule loading by generating a spectral signature of the fluorescent intensity per excitation and emission wavelengths. Comparing metal oxide nanoparticles revealed a unique spectral signature per material composition. 2D FDS showed to be sensitive to changes in surface properties between ZnO NPs synthesized by different methods. ZnO NP loaded with glycol chitosan, polyacrylic acid (PAA), or methoxy polyethylene glycol (mPEG) exhibited a distinct spectral signature shift. ZnO NP loaded with Torula Yeast RNA (TYRNA)(640 nm), polyinosinic: polycytidylic acid (pIC)(680 nm), or splice switching oligonucleotide (SSO)(650 nm) each revealed a shift in emission. Ras-Binding domain (RBD) at three concentrations (25, 37.5, 50 mu g/mL) showed that fluorescent intensity was inversely related to the concentration of protein loaded. These data support 2D FDS as a novel technique in identifying nanoparticles and their surface interactions as a quality assurance tool.
Open Access
Self-Assembly of Amphiphilic Dendrimers: The Role of Generation and Alkyl Chain Length in siRNA Interaction
(2016-07-05) Marquez-Miranda, V.; Araya-Duran, I.; Camarada, M. B.; Comer, Jeffrey; Valencia-Gallegos, J. A.; Gonzalez-Nilo, F. D.; jeffcomer; Comer, Jeffrey
An ideal nucleic-acid transfection system should combine the physical and chemical characteristics of cationic lipids and linear polymers to decrease cytotoxicity and uptake limitations. Previous research described new types of carriers termed amphiphilic dendrimers (ADs), which are based on polyamidoamine dendrimers (PAMAM). These ADs display the cell membrane affinity advantage of lipids and preserve the high affinity for DNA possessed by cationic dendrimers. These lipid/dendrimer hybrids consist of a low-generation, hydrophilic dendron (G2, G1, or G0) bonded to a hydrophobic tail. The G2-18C AD was reported to be an efficient siRNA vector with significant gene silencing. However, shorter tail ADs (G2-15C and G2-13C) and lower generation (G0 and G1) dendrimers failed as transfection carriers. To date, the self-assembly phenomenon of this class of amphiphilic dendrimers has not been molecularly explored using molecular simulation methods. To gain insight into these systems, the present study used coarse-grained molecular dynamics simulations to describe how ADs are able to self-assemble into an aggregate, and, specifically, how tail length and generation play a key role in this event. Finally, explanations are given for the better efficiency of G2/18-C as gene carrier in terms of binding of siRNA. This knowledge could be relevant for the design of novel, safer ADs with well-optimized affinity for siRNA.
Open Access
Expansion of amphibian intronless interferons revises the paradigm for interferon evolution and functional diversity
(2016-06-30) Sang, Y. M.; Liu, Qinfang; Lee, Jinhwa; Ma, Wenjun; McVey, D. Scott; Blecha, Frank; ysang; wjma; blecha; Sang, Yongming; Ma, Wenjun; Blecha, Frank; Liu, Qinfang; McVey, D. Scott
Interferons (IFNs) are key cytokines identified in vertebrates and evolutionary dominance of intronless IFN genes in amniotes is a signature event in IFN evolution. For the first time, we show that the emergence and expansion of intronless IFN genes is evident in amphibians, shown by 24-37 intronless IFN genes in each frog species. Amphibian IFNs represent a molecular complex more complicated than those in other vertebrate species, which revises the established model of IFN evolution to facilitate re-inspection of IFN molecular and functional diversity. We identified these intronless amphibian IFNs and their intron-containing progenitors, and functionally characterized constitutive and inductive expression and antimicrobial roles in infections caused by zoonotic pathogens, such as influenza viruses and Listeria monocytogenes. Amphibians, therefore, may serve as overlooked vectors/hosts for zoonotic pathogens, and the amphibian IFN system provides a model to study IFN evolution in molecular and functional diversity in coping with dramatic environmental changes during terrestrial adaption.
Open Access
Manufacturing Cells for Clinical Use
Weiss, Mark L.; Rao, M. S.; Deans, R.; Czermak, Peter; mlweiss; pczermak; Weiss, Mark L.; Czermak, Peter
The growth in the number of registered clinical trials indicates that there is a need for cells for many types of cell therapy. Figure 1, which is reprinted from the excellent blog maintained by Alexi Bersenev, shows that the cell type used in most clinical trials worldwide is the mesenchymal stromal cell (MSC). The MSC type requires in vitro expansion to reach a clinical dose and thus there is a desire to optimize and standardize processes and procedures for MSC manufacture specifically for clinical use.
Open Access
Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Nsp4 Cleaves VISA to Impair Antiviral Responses Mediated by RIG-I-like Receptors
(2016-06-22) Huang, C.; Du, Y. P.; Yu, Z. B.; Zhang, Q.; Liu, Y. H.; Tang, J.; Shi, Jishu; Feng, W. H.; jshi; Shi, Jishu
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most significant etiological agents in the swine industry worldwide. It has been reported that PRRSV infection can modulate host immune responses, and innate immune evasion is thought to play a vital role in PRRSV pathogenesis. In this study, we demonstrated that highly pathogenic PRRSV (HP-PRRSV) infection specifically down-regulated virus-induced signaling adaptor (VISA), a unique adaptor molecule that is essential for retinoic acid induced gene-I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) signal transduction. Moreover, we verified that nsp4 inhibited IRF3 activation induced by signaling molecules, including RIG-I, MDA5, VISA, and TBK1, but not IRF3. Subsequently, we demonstrated that HP-PRRSV nsp4 down-regulated VISA and suppressed type I IFN induction. Importantly, VISA was cleaved by nsp4 and released from mitochondrial membrane, which interrupted the downstream signaling of VISA. However, catalytically inactive mutant of nsp4 abolished its ability to cleave VISA. Interestingly, nsp4 of typical PRRSV strain CH-1a had no effect on VISA. Taken together, these findings reveal a strategy evolved by HP-PRRSV to counteract anti-viral innate immune signaling, which complements the known PRRSV-mediated immune-evasion mechanisms.