Overlay Assay Research Articles

Long-Term Effect against Methicillin-Resistant Staphylococcus aureus of Emodin Released from Coaxial Electrospinning Nanofiber Membranes with a Biphasic Profile.

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious and rapidly growing threat to human beings. Emodin has a potent activity against MRSA; however, its usage is limited due to high hydrophobicity and low oral bioavailability. Thus, the coaxial electrospinning nanofibers encapsulating emodin in the core of hydrophilic poly (vinylpyrrolidone), with a hygroscopic cellulose acetate sheath, have been fabricated to provide long-term effect against MRSA. Scanning electron microscopy and transmission electron microscopy confirmed the nanofibers had a linear morphology with nanometer in diameter, smooth surface, and core-shell structure. Attenuated total reflection-Fourier transform infrared spectra, X-ray diffraction patterns, and differential scanning calorimetric analyses verified emodin existed in amorphous form in the nanofibers. The nanofibers have 99.38 ± 1.00% entrapment efficiency of emodin and 167.8 ± 0.20% swelling ratio. Emodin released from nanofibers showed a biphasic drug release profile with an initial rapid release followed by a slower sustained release. CCK-8 assays confirmed the nontoxic nature of the emodin-loaded nanofibers to HaCaT cells. The anti-MRSA activity of the nanofibers can persist up to 9 days in AATCC147 and soft-agar overlay assays. These findings suggest that the emodin-loaded electrospun nanofibers with core-shell structure could be used as topical drug delivery system for wound infected by MRSA.

Myoviridae phage PDX kills enteroaggregative Escherichia coli without human microbiome dysbiosis.

Introduction Bacteriophage therapy can be developed to target emerging diarrhoeal pathogens, but doing so in the absence of microbiome disruption, which occurs with antibiotic treatment, has not been established.Aim Identify a therapeutic bacteriophage that kills diarrhoeagenic enteroaggregative Escherichia coli (EAEC) while leaving the human microbiome intact.Methodology Phages from wastewater in Portland, OR, USA were screened for bacteriolytic activity by overlay assay. One isolated phage, PDX, was classified by electron microscopy and genome sequencing. A mouse model of infection determined whether the phage was therapeutic against EAEC. 16S metagenomic analysis of anaerobic cultures determined whether a normal human microbiome was altered by treatment.Results Escherichia virus PDX, a member of the strictly lytic family Myoviridae, killed a case-associated EAEC isolate from a child in rural Tennessee in a dose-dependent manner, and killed EAEC isolates from Columbian children. A single dose of PDX (multiplicity of infection: 100) 1 day post-infection reduced EAEC recovered from mouse faeces. PDX also killed EAEC when cultured anaerobically in the presence of human faecal bacteria. While the addition of EAEC reduced the β-diversity of the human microbiota, that of the cultures with either faeces alone, faeces with EAEC and PDX, or with just PDX phage was not different statistically.Conclusion PDX killed EAEC isolate EN1E-0007 in vivo and in vitro, while not altering the diversity of normal human microbiota in anaerobic culture, and thus could be part of an effective therapy for children in developing countries and those suffering from EAEC-mediated traveller’s diarrhoea without causing dysbiosis.

New bacteriocin-like substances produced by Streptomyces species with activity against pathogens.

Streptomyces spp. are Gram-positive bacteria well-known for their ability to produce antibiotics and other metabolites, but few studies on bacteriocins produced by these bacteria have been reported. We tested eight Streptomyces strains against different pathogenic bacteria, and selected S. griseus, S. nigrescens, S. bottroprensis, and S. violaceoruber for further study based on their inhibitory effects against bacteria, including human pathogens. S. bottropensis reached its highest activity at 312h and was higher than the activities of S. violaceoruber and S. nigrescens. The best condition for bacteriocin precipitation was using diammonium sulfate at 50% saturation. Bacteriocins were susceptible to proteinase treatments and stable at high temperature (up to 100°C). The highest inhibitory activities were observed between pH5 and 6. Cross-activity assays indicated that each Streptomyces strain produced different bacteriocins. When preparations of S. griseus and S. nigrescens were subjected to SDS-PAGE, bands of inhibition were observed in the gel overlay assay at a position corresponding to ~ 2 and 3kDa, respectively, suggesting that both strains are potential sources for novel bacteriocins.

Antifungal effects of Lactobacillus acidophilus and Lactobacillus plantarum against different oral Candida species isolated from HIV/ AIDS patients: an in vitro study

ABSTRACT Oropharyngeal Candidiasis (OPC) is an opportunistic fungal infection occurring in immunocompromised patients such as HIV/AIDS. The purpose of this study was to evaluate the antifungal properties of Lactobacillus acidophilus and Lactobacillus plantarum on different Candida species isolated from oral cavity of HIV/AIDS patients compared to Fluconazole (FLC). In this study, the antifungal effects of both cells and cell-free supernatants (CFSs) of L. acidophilus and L. plantarum were investigated against different oral Candida species by co-aggregation, agar overlay interference and broth microdilution assays, respectively. Our results showed that the highest co-aggregation ratio of the two tested Lactic acid bacteria (LAB) was observed for C. krusei. Both L. acidophilus and L. plantarum at cell concentrations 1010 to 102 cfu/ml were able to inhibit the growth of most of the oral Candida species, except for C. albicans, and to some C. krusei. In this study, MIC and MFC values for CFS of L. acidophilus ranged from 100 to 200 µl/ml and 100 to 200 µl/ml, respectively, and MIC and MFC values for CFS of L. plantarum were 50 to 200 µl/ml and 50 to 200 µl/ml, respectively. The ranges of MIC and MFC for FLC were 256–1024 µg/ml and 512–2048 µg/ml, respectively. C. albicans and C. parapsilosis displayed the highest and least susceptibility to CFSs of two LAB, respectively. Our findings showed that both cells and CFSs of L. acidophilus and L. plantarum had antifungal effects against oral Candida species.

Cell-Free Protein Translation System for Expression of Lipid-Binding Proteins Tagged with Small epitopes and Their Use in Protein-Lipid Overlay Assays.

We adapted an efficient cell-free protein synthesis-based protocol for the production of lipid-binding proteins. The experimental procedures are based on the following steps: (1) cell-free synthesis of soluble, lipid-binding proteins fused to small tags; (2) analysis by dot blot of the accessibility of antibodies to the small tags. (3) protein lipid overlay assay with, immunodetection of bound protein by either chemiluminescence or fluorescence. We also provide a fast and inexpensive protocol for homemade lipid nitrocellulose strips spotted with acidic lipids (mostly phosphoinositides) extracted from plant tissues. These homemade lipid strips can be used for preliminary screen and characterization of putative phosphoinositide-binding proteins.

Different Forms of TFF2, A Lectin of the Human Gastric Mucus Barrier: In Vitro Binding Studies

Trefoil factor family 2 (TFF2) and the mucin MUC6 are co-secreted from human gastric and duodenal glands. TFF2 binds MUC6 as a lectin and is a constituent of the gastric mucus. Herein, we investigated human gastric extracts by FPLC and identified mainly high- but also low-molecular-mass forms of TFF2. From the high-molecular-mass forms, TFF2 can be completely released by boiling in SDS or by harsh denaturing extraction. The low-molecular-mass form representing monomeric TFF2 can be washed out in part from gastric mucosa specimens with buffer. Overlay assays with radioactively labeled TFF2 revealed binding to the mucin MUC6 and not MUC5AC. This binding is modulated by Ca2+ and can be blocked by the lectin GSA-II and the monoclonal antibody HIK1083. TFF2 binding was also inhibited by Me-β-Gal, but not the α anomer. Thus, both the α1,4GlcNAc as well as the juxtaperipheral β-galactoside residues of the characteristic GlcNAcα1→4Galβ1→R moiety of human MUC6 are essential for TFF2 binding. Furthermore, there are major differences in the TFF2 binding characteristics when human is compared with the porcine system. Taken together, TFF2 appears to fulfill an important role in stabilizing the inner insoluble gastric mucus barrier layer, particularly by its binding to the mucin MUC6.

Antifungal Potency and Modes of Action of a Novel Olive Tree Defensin Against Closely Related Ascomycete Fungal Pathogens.

Antimicrobial peptides play a pivotal role in the innate immunity of plants. Defensins are cysteine-rich antifungal peptides with multiple modes of action. A novel Oleaceae-specific defensin gene family has been discovered in the genome sequences of wild and cultivated species of a perennial olive tree, Olea europaea. OefDef1.1, a member of this defensin family, potently inhibits the in-vitro growth of ascomycete fungal pathogens Botrytis cinerea and three Fusarium spp. OefDef1.1 rapidly permeabilizes the plasma membrane of the conidial and germling cells of B. cinerea. Interestingly, it induces reactive oxygen species and translocates to the cytoplasm only in the germlings but not in the conidia. In medium containing a high concentration of Na1+, antifungal activity of OefDef1.1 is significantly reduced. Surprisingly, a chimeric OefDef1.1 peptide containing the γ-core motif of a Medicago truncatula defensin, MtDef4, displays Na1+-tolerant antifungal activity. In a phospholipid-protein overlay assay, the chimeric peptide exhibits stronger binding to its phosphoinositide partners than OefDef1.1 and is also more potent in inhibiting gray mold disease on the surface of Nicotiana benthamiana and lettuce leaves than OefDef1.1. Significant differences are observed among the four ascomycete pathogens in their responses to OefDef1.1 in growth medium with or without the elevated concentration of Na1+. The varied responses of closely related ascomycete pathogens to this defensin have implications for engineering disease resistance in plants.

Screening for Antibiotic-Producing Bacteria from Imbak Canyon Conservation Area (ICCA)

Antibiotic resistance is an escalating threat to public health. Therefore, there is an urgent need for new antibiotics. This study aims to screen for antibiotic-producing microorganisms from the forest soil of Batu Timbang. Soil samples were collected, diluted and spread plated onto 1/5 Nutrient Agar (NA) and Actinomycete Isolation Agar (AIA) for the isolation of antibiotic-producing microorganisms. A total of 180 bacterial isolates were screened for their antibiotic-producing ability, and ten were tested positive for inhibitory activity against one or more test pathogens via agar overlay assay (Staphylococcus aureus ATCC BAA-1717, Enterococcus faecalis ATCC 700802, and Acinetobacter baumannii ATCC BAA-1605). Ten bacterial isolates were subjected to 16S rRNA gene amplification and gene sequence analysis. The isolates were identified to be closely related to the genus Variovorax, Streptomyces, Kitasatospora, Chromobacterium, Burkholderia, Pseudomonas and Massilia. Three isolates (Variovorax sp. A5, Variovorax sp. A6 and Kitasatospora sp. H8) are potentially novel as these isolates form a different clade from their respective closely related species via phylogenetic tree analysis using reference sequences obtained from GenBank/EMBL/DDBJ databases. The antibiotics produced by the bacterial isolates might potentially be new, as novel species might possess unique biosynthetic gene clusters to produce new compounds. Nevertheless, further taxonomic identification and antibiotic isolation work is required. This study has revealed the potential of antibiotic discovery from Batu Timbang (Imbak Canyon Conservation Area) and its implications in tackling antibiotic resistance.

Expansion of Hydra actinoporin-like toxin (HALT) gene family: Expression divergence and functional convergence evolved through gene duplication

Hydra actinoporin-like toxin 1 (HALT-1) was previously shown to cause cytolysis and haemolysis in a number of human cells and has similar functional properties to the actinoporins equinatoxin and sticholysin. In addition to HALT-1, five other HALTs (HALTs 2, 3, 4, 6 and 7) were also isolated from Hydra magnipapillata and expressed as recombinant proteins in this study. We demonstrated that recombinant HALTs have cytolytic activity on HeLa cells but each exhibited a different range of toxicity. All six recombinant HALTs bound to sulfatide, while rHALT-1 and rHALT-3 bound to two additional sphingolipids, lysophosphatidic acid and sphingosine-1-phosphate as indicated by the protein-lipid overlay assay. When either tryptophan133 or tyrosine129 of HALT-1 was mutated, the mutant protein lost binding to sulfatide, lysophosphatidic acid and sphingosine-1-phosphate. As further verification of HALTs’ binding to sulfatide, we performed ELISA for each HALT. To determine the cell-type specific gene expression of seven HALTs in Hydra, we searched for individual HALT expression in the single-cell RNA-seq data set of Single Cell Portal. The results showed that HALT-1, 4 and 7 were expressed in differentiating stenoteles. HALT-1 and HALT-6 were expressed in the female germline during oogenesis. HALT-2 was strongly expressed in the gland and mucous cells in the endoderm. Information on HALT-3 and HALT-5 could not be found in the single-cell data set. Our findings show that subfunctionalisation of gene expression following duplication enabled HALTs to become specialized in various cell types of the interstitial cell lineage.

The Evaluation of Antimicrobial and Antibiofilm Activity of Bioactive Compounds Obtained from Aspergillus Sclerotiorum

This study was concerned with the screening of antimicrobial products from fungi collected from soil and evalution of their antibiofilm activity. The isolate having antimicrobial and antibiofilm compounds was characterized by the molecular methods and identified as Aspergillus sclerotiorum. A. sclerotiorum was grown in yeast peptone glucose (YPG) medium and extracellular medium was extraction by 1:1 ethyl acetate. Crude extraction characterized through thin layer chromatography (TLC) on silica gel 60 HF254 and was detected five bands. Agar diffusion and TLC overlay assays were done against Gram-positive (Staphylococcus aureus ATCC 25923, meticilin resistance S. aureus (MRSA) and Enterococcus faecalis ATCC 29212) and Gram-negative bacteria (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853). The most dense band (Rf:0.42) showed the best inhibition zone on TLC overlay. The results showed that the most dense band can potential source for antimicrobial compound. After the most dense band in silica gel was scraped and dissolved ethyl acetate for minimum inhibitory concentration (MIC) determination and crystal violet assay against S. aureus and MRSA. These results indicate that fungi, A. sclerotiorum, isolated from soil was potential source for antimicrobial and antibiofilm compounds.

Breast Cancer Cells Transition from Mesenchymal to Amoeboid Migration in Tunable Three-Dimensional Silk-Collagen Hydrogels.

Invading cancer cells adapt their migration phenotype in response to mechanical and biochemical cues from the extracellular matrix. For instance, mesenchymal migration is associated with strong cell-matrix adhesions and an elongated morphology, while amoeboid migration is associated with minimal cell-matrix adhesions and a rounded morphology. However, it remains challenging to elucidate the role of matrix mechan-ics and biochemistry, since these are both dependent on ECM protein concentration. Here, we demonstrate a composite silk fibroin and collagen I hydrogel where stiffness and microstructure can be systematically tuned over a wide range. Using an overlay assay geometry, we show that the invasion of metastatic breast cancer cells exhibits a biphasic dependence on silk fibroin concentration at fixed collagen I concentration, first increasing as the hydrogel stiffness increases, then decreasing as the pore size of silk fibroin decreases. Indeed, mesenchymal morphology exhibits a similar biphasic depen-dence on silk fibroin concentration, while amoeboid morphologies were favored when cell-matrix adhesions were less effective. We used exogenous biochemical treatment to perturb cells towards increased contractility and a mesenchymal morphology, as well as to disrupt cytoskeletal function and promote an amoeboid morphology. Overall, we envision that this tunable biomaterial platform in a 96-well plate format will be widely applicable to screen cancer cell migration against combinations of designer biomaterials and targeted inhibitors.

Promiscuous Dimerization Between the Caenorhabditis elegans IF Proteins and a Hypothesis to Explain How Multiple IFs Persist Over Evolutionary Time.

Our previous calculations of ionic interactions indicated that the Caenorhabditis elegans intermediate filament (IF) IFA proteins, in addition to IFA/IFB-1 heterodimers, may also form homodimers. In order to prove the significance of these calculations, we analysed the dimerization potential of the IFA chains in blot overlays. Unexpectedly, we found here that the dimerization of the IFA-1 protein was of both homotypic and heterotypic nature, and involved all proteins immobilized on the membrane (IFA-1, IFA-2, IFA-4, IFB-1, IFB-2, IFC-1, IFC-2, IFD-1, IFD-2 and IFP-1). A similar interaction profile, though less complex, was observed for two biotinylated proteins (IFA-2 and IFA-4). These and previous results indicate that the IFA proteins are able to form many different heteropolymeric and homopolymeric complexes in the C. elegans tissue, but that only those triggered by the IFA-specific IFB-1 protein result in mature IFs. Moreover, the calculations of the possible ionic interactions between the individual rod sequences as well as their various deletion variants indicated a special role in this process for the middle part of the C. elegans IF coil 1B segment that is deleted in all vertebrate cytoplasmic IFs. We hypothesized here, therefore, that the striking promiscuity of the C. elegans IFs originally involved a nuclear lamin which, due to a two-heptad-long rod deletion, prevented formation of a functional lamin/cIF dimer. This, in concert with an efficient dimerization and a strict tissue-specific co-expression, may allow expansion and maintenance of the multiple Caenorhabditis IFs. A possible implication for evolution of chordate IFs proteins is also discussed.

Chumacin-1 and Chumacin-2 from Pseudomonas aeruginosa strain CGK-KS-1 as novel quorum sensing signaling inhibitors for biocontrol of bacterial blight of rice

The in vitro inhibition of quorum sensing signal, xanthan gum secretion, biofilm formation in different Xanthomonas pathovars and biological control of bacterial blight of rice by the two bioactive extrolites produced by Pseudomonas aeruginosa strain CGK-KS-1 were explored. These extrolites were extracted from Diaion HP-20 resin with methanol and purified by preparative-thin layer chromatography. Further, spectroscopic structural elucidation revealed the tentative identity of these extrolites to be (R,3E,5E,9Z,11E)-13-((3S,5R)-5-acetyl-2,6-dimethylheptan-3-yl)-10-hydroxy-4-methyl-1,8-diazabicyclo[9.3.1]pentadeca-3,5,9,11(15),13-pentaen-2-one and (R,3E,5E,8E,11E)-13-((3S,5R)-5-acetyl-2,6-dimethylheptan-3-yl)-4-methyl-1,8-diazabicyclo[9.3.1]pentadeca-3,5,8,11(15),13-pentaene-2,10-dione, named as Chumacin-1 and Chumacin-2, respectively. Antimicrobial assay showed Chumacin-1 and Chumacin-2 exhibited a strong in vitro growth inhibition against various Xanthomonas pathovars. Quorum sensing overlay assay using a reporter strain Chromobacterium violaceum strain CV026 showed that Chumacin-1 and Chumacin-2 inhibited quorum sensing signaling. The mechanistic studies revealed that these extrolites inhibited the production of quorum sensing signaling factor, cis-11-methyl-2-dodecenoic acid; suppressed the xanthan gum secretion and also inhibited the biofilms formed by various Xanthomonas pathovars. Both Chumacin-1 and Chumacin-2 showed ROS generation in the test Xanthomonas strains, resulting in in vitro cell membrane damage was revealed through CSLM and FE-SEM micrographs. Further, greenhouse experiments using Samba Mashuri (BPT-5204) revealed that seed treatment with Chumacin-1 and Chumacin-2 along with foliar spray groups showed up to ˜80% reduction in bacterial blight disease in rice. To the best of our knowledge, this is the first report on new quorum sensing inhibitors, Chumacin-1 and Chumacin-2 produced by Pseudomonas aeruginosa strain CGK-KS-1 exhibiting DSF inhibition activity in Xanthomonas oryzae pv. oryzae.

Short communication: Screening inhibition of dairy-relevant pathogens and spoilage microorganisms by lactose oxidase

The inhibitory effect of lactose oxidase on the growth of foodborne pathogens and spoilage microorganisms associated with dairy products was evaluated through an overlay inhibition assay. Lactose oxidase generates hydrogen peroxide via lactose oxidation into lactobionic acid. Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica ser. Typhimurium, Staphylococcus aureus, Pseudomonas fragi, and Penicillium chrysogenum were used as indicators. A commercially available solution of lactose oxidase was applied at different concentrations (0, 0.12, 1.2, and 12 g/L) in 4 types of media [brain heart infusion agar (BHI), BHI + sodium thiocyanate (NaSCN), BHI + lactose, and BHI + NaSCN + lactose] to evaluate the effect of lactose and thiocyanate on microbial inhibition. Lactose oxidase inhibited the growth of all the indicators at a concentration of 12 g/L of the enzyme solution in the presence of lactose alone and in combination with NaSCN. However, supplementation with NaSCN had no effect on the magnitude of microbial inhibition. Staphylococcus aureus was the most sensitive pathogen, and Ps. fragi was the most sensitive of all the indicators in general to lactose oxidase. Listeria monocytogenes and Ps. fragi showed higher susceptibility to the antimicrobial effect of lactose oxidase at 6°C than at their corresponding optimum growth temperature. The inhibitory effect was attributed to the generation of hydrogen peroxide from the oxidation of lactose. Findings from this study demonstrate that lactose oxidase could be used as a novel approach to inhibit the growth of mold and bacteria. It could also be applied as a label-friendly preservative in dairy foods.

ISOLATION OF PHAGES INFECTING LISTERIA MONOCYTOGENES

It was aimed to isolate, purify and determine host ranges of lytic phages that infect Listeria monocytogenes in this study. Out of 68 samples screened, 4 positive isolates were recovered from feces, food processing waste water, fisheries waste water, and fish samples. Recovery status of the L. monocytogenes phage was found to be 5.88%. To determine host ranges of phages, soft agar overlay plaque assay was used. While eleven L. monocytogenes strains showed resistant to all four isolated phages, 24 strains were sensitive. The plaque sizes of the 4 phages against 24 L. monocytogenes strains ranged from 0.4 and 2.6 mm. None of the phages had identical host ranges.

Brevibacillus laterosporus strains BGSP7, BGSP9 and BGSP11 isolated from silage produce broad spectrum multi-antimicrobials.

Bacteria active against multi-drug resistant pathogens, isolated by direct selection of colonies from clover silage samples, produce zones of inhibition against two Gram-negative (Klebsiella pneumoniae Ni9 and Pseudomonas aeruginosa MMA83) and two Gram-positive (Staphylococcus aureus ATCC25923 and Listeria monocytogenes ATCC19111) pathogens. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 produced the largest zones of inhibition against all four pathogens when grown in LB broth with aeration at 37°C. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 were identified as Brevibacillus laterosporus and pulsed field gel electrophoresis and extracellular protein profiles showed that three different strains (BGSP7, BGSP9 and BGSP11) were isolated. A semi-native SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel overlay assay showed that BGSP7 and BGSP9 produce small antimicrobial molecules of about 1.5 kDa, while BGSP11 produces antimicrobial molecules of 1.5 and 6 kDa active against S. aureus ATCC25923. Amino acid analysis of two antimicrobial molecules (1583.73 Da; from BGSP7 and 1556.31 Da; from BGSP11) revealed that they have a similar composition and differ only by virtue of the presence of a methionine which is present only in BGSP11 molecule. Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides (brevibacillin, bogorol, gramicidin S, plipastatin and tyrocin) and bacteriocins (laterosporulin, a lactococcin 972-like bacteriocin, as well as putative linocin M18, sactipeptide, UviB and lantipeptide-like molecules). Ultimately, the purification of a number of antimicrobial molecules from each isolate suggests that they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects.

Annexin II as a Dengue Virus Serotype 2 Interacting Protein Mediating Virus Interaction on Vero Cells.

Recent evidence has demonstrated that dengue virus requires active filopodia formation for a successful infection. However, the cellular factor involved in the interaction has not been fully elucidated. We used a combination of virus overlay protein binding assay and LC-MS/MS, and identified annexin II as a dengue virus serotype 2 (DENV2) interacting protein on Vero cells, upon filopodia induction. Flow cytometry analysis showed annexin II on the Vero cells surface increased when DENV2 was added. The amount of annexin II in the plasma membrane fraction was reduced as the infection progressed. Antibody-mediated inhibition of infection and siRNA-mediated knockdown of annexin II expression significantly reduced DENV2 infection and production levels. Collectively, we demonstrated that annexin II is one of the host factor involved in DENV2 binding on Vero cells.

Ezrin is a novel target in cyst initiation in ADPKD

Autosomal dominant polycystic kidney disease (ADPKD), characterized by progressive renal cystogenesis and other extrarenal manifestations, is one of the most common monogenetic diseases, and is caused by loss of function mutations in PKD1 and PKD2, which encode transmembrane proteins polycystin 1 and 2 (PC1/2). The polycystin proteins have been hypothesized to function as a complex that has been localized to the plasma membrane, cilia, endoplasmic reticulum, and junctions of epithelial cells. The downstream effects of either PC1 or PC2 function loss have not been elucidated. Previously, an investigation of smaller cysts from nephrectomized human ADPKD kidneys revealed a pattern of increased cell width and an altered apical compartment. Subsequently, we observed a decreased abundance and disrupted localization of ezrin, a master scaffold and apical compartment regulator, in similarly characterized cysts. Following this observation, we explored the role of ezrin disruption in a three‐dimensional in vitro model of cystogenesis. Acute inactivation of Pkd2 lead to significant loss of ezrin protein. We confirmed the regulatory relationship between PC2 and ezrin with immunohistochemistry in a genetically inducible in vivo short induction model of Pkd2 inactivation (Pkd2fl/fl Pax8 rtTA TetOCre) that revealed a kidney tubule wide decrease in ezrin at the apical membrane. Lipid protein overlay assays and immunoprecipitation experiments demonstrate both PC2 and ezrin interact with similar phosphoinositides, and possibly with each other, suggesting a role for PC2 in apical compartment and junctional assembly. Functionally, we found that PC2 is critical in junctional integrity, with acute inactivation of Pkd2 causing decreases in transepithelial electrical resistance in inducible primary renal epithelial cells (Pkd2fl/fl Pax8 rtTA TetOCre). Ezrin, expressed in all segments of the kidney, is a regulator of many components of the junctions and apical compartment of renal epithelial cells. The disruption of ezrin following inactivation of Pkd2 in both in vitro and in vivo models, and shared interacting lipids, suggests that PC2 may play a role in the regulation of ezrin in renal epithelial cells and that ezrin may be involved in the initiation of cystogenesis following loss of PC2.Support or Funding InformationNIDDK F31 NRSA, PKD Foundation, NIDDK Baltimore Polycystic Kidney Disease Research and Clinical Core CenterThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Understanding the mechanism of Chikungunya virus vector competence in three species of mosquitoes.

Chikungunya virus (CHIKV) is primarily transmitted by Aedes spp. mosquitoes. The present study investigated vector competence for CHIKV in Aedes aegypti and Aedes albopictus mosquitoes found in Madurai, South India. The role of receptor proteins on midguts contributing to permissiveness of CHIKV to Aedes spp. mosquitoes was also undertaken. Mosquitoes were orally infected with CHIKV DRDE-06. Infection of midguts and dissemination to heads was confirmed by immunofluorescence assay at different time points. A plaque assay was performed from mosquito homogenates at different time points to study CHIKV replication. Presence of putative CHIKV receptor proteins on mosquito midgut epithelial cells was detected by virus overlay protein binding assay (VOPBA). The identity of these proteins was established using mass spectrometry. CHIKV infection of Ae. aegypti and Ae. albopictus midguts and dissemination to heads was observed to be similar. A plaque assay performed with infected mosquito homogenates revealed that CHIKV replication dynamics was similar in Aedes sp. mosquitoes until 28 days post infection. VOPBA performed with mosquito midgut membrane proteins revealed that prohibitin could serve as a putative CHIKV receptor on Aedes mosquito midguts, whereas an absence of CHIKV binding protein/s on Culex quinquefasciatus midguts can partially explain the non-permissiveness of these mosquitoes to infection.

Comparative Activity of Commercial Baculovirus Formulations against Heliothine Pests of Cotton

Nuclear polyhedrosis viruses have been labeled for use as microbial insecticides in agronomically-important crops in the U.S. since the 1970s. New products developed from viral isolates of different species in the heliothine genera Helicoverpa, Heliothis, and Chloridea have been introduced to the market and are receiving renewed attention for controlling heliothine pests. Two laboratory assays were used to evaluate comparative activities among the first nuclear polyhedrosis viruses registered in the U.S. and two commercially-available baculovirus products for controlling bollworm, Helicoverpa zea (Boddie), and tobacco budworm, Chloridea virescens (F.). Based on comparisons of LC50s of formulated products from laboratory overlay assays at 7 days, Gemstar was 2.4 and 3.7 fold less active than Elcar against bollworm and tobacco budworm, respectively. Heligen was 2.9 and 7.1 fold more active than Elcar against bollworm and tobacco budworm, respectively. However, once corrected for concentration of polyhedral occlusion bodies, susceptibility of bollworm did not differ to any of the three products tested. Tobacco budworms were more susceptible to Heligen than Gemstar in laboratory overlay assays following correction for differences in concentration of polyhedral occlusion bodies, which might be caused by interrelatedness of the species with the Old World bollworm. No discernable difference in death of bollworms or tobacco budworms was detected among the greatest labeled rate of any formulated microbial insecticide sprayed on leaves of non-Bt cotton (Gossypium hirsutum L.) at 14 days and percentage of pupation at 20 days post-treatment. Although the activity of wild-type baculovirus has changed little during the past four decades, use of the products should be encouraged whenever possible. Potential ecological benefits of preserving beneficial insects and reducing selection for resistance to insecticides should be promoted as long as effectiveness and costs are reasonable.