Plasmids Expressing shRNAs Specific to the Nucleocapsid Gene Inhibit the Replication of Porcine Deltacoronavirus In Vivo

Porcine deltacoronavirus (PDCoV) is a novel enteric coronavirus and is becoming one of the major causative agents of diarrhea in pig herds in recent years. To date, there are no commercial vaccines or antiviral pharmaceutical agents available to control PDCoV infection. Therefore, developing a reliable strategy against PDCoV is urgently needed. In this study, to observe the antiviral activity of RNA interference (RNAi), four short hairpin RNAs (shRNAs) specific to the nucleocapsid (N) gene of PDCoV were designed and tested in vitro. Of these, a double-shRNA-expression vector, designated as pSil-double-shRNA-N1, was the most effectively expressed, and the inhibition of PDCoV replication was then further evaluated in neonatal piglets. Our preliminary results reveal that plasmid-based double-shRNA-expression targeting the N gene of PDCoV can significantly protect LLC-PK1 cells and piglets from pathological lesions induced by PDCoV. Our study could benefit the investigation of the specific functions of viral genes related to PDCoV infection and offer a possible methodology of RNAi-based therapeutics for PDCoV infection.

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Adenovirus-Mediated In Vivo Silencing of Anaphylatoxin Receptor C5aR

Journal of Biomedicine and Biotechnology ◽

10.1155/jbb/2006/28945 ◽

2006 ◽

Vol 2006 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Lei Sun ◽

Hongwei Gao ◽

Vidya J. Sarma ◽

Ren-feng Guo ◽

Peter A. Ward

Keyword(s):

G Protein Coupled Receptors ◽

Mouse Lung ◽

Transcriptional Level ◽

Shrna Expression ◽

C5a Receptor ◽

Immunological Diseases ◽

Short Hairpin Rnas ◽

G Protein Coupled

C5a, one of the most potent inflammatory peptides, induces its inflammatory functions by interacting with C5a receptor (C5aR) that belongs to the rhodopsin family of seven-transmembrane G protein-coupled receptors. C5a/C5aR signaling has been implicated in the pathogenesis of many inflammatory and immunological diseases such as sepsis and acute lung injury. Widespread upregulation of C5aR has been seen at both the protein level and transcriptional level under pathological conditions. Here, we show that C5aR gene expression can be specifically suppressed by siRNA, both in vitro and in vivo. A panel of chemically siRNA oligonucleotides was first synthesized to identify the functional siRNA sequences. The short hairpin RNAs (shRNAs) were also designed, cloned, and tested for the silencing effects in C5aR transfected cells. The effective shRNA expression cassettes were then transferred to an adenovirus DNA vector. ShRNA-expressing adenoviruses were intratracheally administered into mouse lung, and a significant in vivo silencing of C5aR was obtained four days after administration. Thus, C5aR shRNA-expressing adenoviruses appear to be an alternative strategy for the treatment of complement-induced disorders.

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Significant Interference with Porcine Epidemic Diarrhea Virus Pandemic and Classical Strain Replication in Small-Intestine Epithelial Cells Using an shRNA Expression Vector

Vaccines ◽

10.3390/vaccines7040173 ◽

2019 ◽

Vol 7 (4) ◽

pp. 173

Author(s):

Da Shi ◽

Xiaobo Wang ◽

Hongyan Shi ◽

Jiyu Zhang ◽

Yuru Han ◽

...

Keyword(s):

Epithelial Cells ◽

Porcine Epidemic Diarrhea Virus ◽

Large Scale ◽

N Gene ◽

Shrna Expression ◽

Diarrhea Virus ◽

Shrna Expression Vector ◽

Wide Range ◽

Porcine Epidemic Diarrhea

Porcine epidemic diarrhea (PED) re-emerged in China in 2010 and is now widespread. Evidence indicates that highly virulent porcine epidemic diarrhea virus (PEDV) strains belonging to genotype G2 caused a large-scale outbreak of diarrhea. Currently, vaccines derived from PEDV classical strains do not effectively prevent infection by virulent PEDV strains, and no specific drug is available to treat the disease. RNA interference (RNAi) is a novel and effective way to cure a wide range of viruses. We constructed three short hairpin RNA (shRNA)-expressing plasmids (shR-N307, shR-N463, and shR-N1071) directed against nucleocapsid (N) and determined their antiviral activities in intestine epithelial cells infected with a classical CV777 strain and LNCT2. We verified that shR-N307, shR-N463, and shR-N1071 effectively inhibited the expression of the transfected N gene in vitro, comparable to the control shRNA. We further demonstrated the shRNAs markedly reduced PEDV CV777 and LNCT2 replication upon downregulation of N production. Therefore, this study provides a new strategy for the design of antiviral methods against coronaviruses by targeting their processivity factors.

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Bovine Milk-Derived Emulsifiers Increase Triglyceride Absorption in Newborn Formula-Fed Pigs

Nutrients ◽

10.3390/nu13020410 ◽

2021 ◽

Vol 13 (2) ◽

pp. 410 ◽

Cited By ~ 1

Author(s):

Kristine Bach Korsholm Knudsen ◽

Christine Heerup ◽

Tine Røngaard Stange Jensen ◽

Xiaolu Geng ◽

Nikolaj Drachmann ◽

...

Keyword(s):

Milk Fat ◽

Bovine Milk ◽

In Vitro Digestion ◽

Lipid Absorption ◽

Lipid Digestion ◽

Soy Lecithin ◽

Neonatal Piglets ◽

Milk Fat Globule Membranes

Efficient lipid digestion in formula-fed infants is required to ensure the availability of fatty acids for normal organ development. Previous studies suggest that the efficiency of lipid digestion may depend on whether lipids are emulsified with soy lecithin or fractions derived from bovine milk. This study, therefore, aimed to determine whether emulsification with bovine milk-derived emulsifiers or soy lecithin (SL) influenced lipid digestion in vitro and in vivo. Lipid digestibility was determined in vitro in oil-in-water emulsions using four different milk-derived emulsifiers or SL, and the ultrastructural appearance of the emulsions was assessed using electron microscopy. Subsequently, selected emulsions were added to a base diet and fed to preterm neonatal piglets. Initially, preterm pigs equipped with an ileostomy were fed experimental formulas for seven days and stoma output was collected quantitatively. Next, lipid absorption kinetics was studied in preterm pigs given pure emulsions. Finally, complete formulas with different emulsions were fed for four days, and the post-bolus plasma triglyceride level was determined. Milk-derived emulsifiers (containing protein and phospholipids from milk fat globule membranes and extracellular vesicles) showed increased effects on fat digestion compared to SL in an in vitro digestion model. Further, milk-derived emulsifiers significantly increased the digestion of triglyceride in the preterm piglet model compared with SL. Ultra-structural images indicated a more regular and smooth surface of fat droplets emulsified with milk-derived emulsifiers relative to SL. We conclude that, relative to SL, milk-derived emulsifiers lead to a different surface ultrastructure on the lipid droplets, and increase lipid digestion.

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Preclinical Studies in Anti-Trypanosomatidae Drug Development

Pharmaceuticals ◽

10.3390/ph14070644 ◽

2021 ◽

Vol 14 (7) ◽

pp. 644

Author(s):

Cintya Perdomo ◽

Elena Aguilera ◽

Ileana Corvo ◽

Paula Faral-Tello ◽

Elva Serna ◽

...

Keyword(s):

Rural Communities ◽

Chagas Disease ◽

Murine Model ◽

Mammalian Cells ◽

Drug Candidate ◽

Murine Macrophages ◽

Causative Agents ◽

Trypanosomatid Parasites

The trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania are the causative agents of human African trypanosomiasis, Chagas Disease and Leishmaniasis, respectively. These infections primarily affect poor, rural communities in the developing world, and are responsible for trapping sufferers and their families in a disease/poverty cycle. The development of new chemotherapies is a priority given that existing drug treatments are problematic. In our search for novel anti-trypanosomatid agents, we assess the growth-inhibitory properties of >450 compounds from in-house and/or “Pathogen Box” (PBox) libraries against L. infantum, L. amazonensis, L.braziliensis, T. cruzi and T. brucei and evaluate the toxicities of the most promising agents towards murine macrophages. Screens using the in-house series identified 17 structures with activity against and selective toward Leishmania: Compounds displayed 50% inhibitory concentrations between 0.09 and 25 μM and had selectivity index values >10. For the PBox library, ~20% of chemicals exhibited anti-parasitic properties including five structures whose activity against L. infantum had not been reported before. These five compounds displayed no toxicity towards murine macrophages over the range tested with three being active in an in vivo murine model of the cutaneous disease, with 100% survival of infected animals. Additionally, the oral combination of three of them in the in vivo Chagas disease murine model demonstrated full control of the parasitemia. Interestingly, phenotyping revealed that the reference strain responds differently to the five PBox-derived chemicals relative to parasites isolated from a dog. Together, our data identified one drug candidate that displays activity against Leishmania and other Trypanosomatidae in vitro and in vivo, while exhibiting low toxicity to cultured mammalian cells and low in vivo acute toxicity.

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Visualization of SARS-CoV-2 infection dynamic

10.1101/2021.06.03.446942 ◽

2021 ◽

Author(s):

Chengjin Ye ◽

Kevin Chiem ◽

Jun-Gyu Park ◽

Jesus Silvas ◽

Desarey Morales Vasquez ◽

...

Keyword(s):

Viral Infection ◽

Cultured Cells ◽

Imaging System ◽

Reporter Genes ◽

Therapeutic Interventions ◽

Wild Type Virus ◽

Nucleocapsid Gene ◽

Recombinant Viruses

Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing rSARS-CoV-2 have jeopardized their use to monitor the dynamics of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the viral nucleocapsid gene followed by a 2A cleavage peptide. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and K18 hACE2 transgenic mice. Importantly, real-time viral infection was readily tracked using a non-invasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2 retained wild-type virus like pathogenicity in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

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Kinetics of Targeted Phage Rescue in a Mouse Model of SystemicEscherichia coliK1

BioMed Research International ◽

10.1155/2018/7569645 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 5

Author(s):

György Schneider ◽

Nikolett Szentes ◽

Marianna Horváth ◽

Ágnes Dorn ◽

Alysia Cox ◽

...

Keyword(s):

Capsular Polysaccharide ◽

Protective Efficacy ◽

Lytic Bacteriophage ◽

E Coli ◽

Causative Agents ◽

High Level ◽

Systemic Infections ◽

Kinetics Of

Escherichia (E.) coliK1 strains remain common causative agents of neonatal sepsis and meningitis. We have isolated a lytic bacteriophage (ΦIK1) againstE. colistrain IHE3034 and tested its specificityin vitro, as well as distribution and protective efficacyin vivo. The phage was shown to be specific to the K1 capsular polysaccharide. In the lethal murine model, a high level of protection was afforded by the phage with strict kinetics. A single dose of 1 x 108phage particles administered 10 and 60 minutes following the bacterial challenge elicited 100 % and 95 % survival, respectively. No mice could be rescued if phage administration occurred 3 hours postinfection. Tissue distribution surveys in the surviving mice revealed that the spleen was the primary organ in which accumulation of active ΦIK1 phages could be detected two weeks after phage administration. These results suggest that bacteriophages have potential as therapeutic agents in the control of systemic infections.

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In Vitro and In Vivo Effectiveness of Carvacrol, Thymol and Linalool against Leishmania infantum

Molecules ◽

10.3390/molecules24112072 ◽

2019 ◽

Vol 24 (11) ◽

pp. 2072 ◽

Cited By ~ 11

Author(s):

Mohammad Reza Youssefi ◽

Elham Moghaddas ◽

Mohaddeseh Abouhosseini Tabari ◽

Ali Akbar Moghadamnia ◽

Seyed Mohammad Hosseini ◽

...

Keyword(s):

Mtt Assay ◽

Leishmania Infantum ◽

Standard Drug ◽

In Vivo Test ◽

Causative Agents ◽

Ic50 Values ◽

Vector Borne ◽

Effective Drugs

Background: One of the most important causative agents of visceral leishmaniasis (VL) is Leishmania infantum, which is mainly spread by Phlebotomus and Lutzomyia sandflies in the Old and New World, respectively. Novel and effective drugs to manage this neglected vector-borne disease are urgently required. In this study, we evaluated the toxicity of carvacrol, thymol and linalool, three common essential oil constituents, on amastigotes and promastigotes of L. infantum. Methods: in vitro experiments were performed by 24 h MTT assay. Carvacrol, thymol and linalool at concentrations ranging from 1.3 to 10 μg/mL were tested on promastigotes of L. infantum. For in vivo test, two groups of hamsters (Mesocricetus auratus) received 100 mg/kg of body weight/day of carvacrol and thymol as intraperitoneal injection on day 7 post-infection, followed by a 48 h later injection. The third group was treated with the glucantime as standard drug (500 mg/kg) and the last group (control) just received normal saline. On the 16th day, the number of parasites and histopathological changes in liver and spleen were investigated. Results: 24 h MTT assay showed promising antileishmanial activity of thymol and carvacrol, with IC50 values of 7.2 (48 μM) and 9.8 μg/mL (65 μM), respectively. Linalool at all concentrations did not affect L. infantum promastigote viability. In vivo toxicity data of carvacrol and thymol showed that the former at 100 mg/kg was the safest and most effective treatment with little side effects on the liver. Conclusions: Overall, thymol and carvacrol are highly promising candidates for the development of effective and safe drugs in the fight against VL.

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Immunological investigations of oligoethylene glycols functionalized with desaminotyrosine and desaminotyrosyltyrosine

MRS Proceedings ◽

10.1557/opl.2013.831 ◽

2013 ◽

Vol 1569 ◽

pp. 9-14 ◽

Cited By ~ 2

Author(s):

Konstanze K. Julich-Gruner ◽

Toralf Roch ◽

Nan Ma ◽

Axel T. Neffe ◽

Andreas Lendlein

Keyword(s):

Human Blood ◽

High Concentration ◽

Inflammatory Conditions ◽

Tnf Α ◽

High Concentrations ◽

Pharmaceutical Agents ◽

Immunological Evaluation ◽

Necrosis Factor

ABSTRACTBiomaterials require thorough in vitro testing before being applied in vivo. Unwanted contaminations of biomaterials but also their intrinsic properties can cause uncontrolled immune response leading to severe consequences for the patient. Therefore, immunological evaluation of materials for biomedical applications is mandatory before entering clinical application. In order to introduce physical netpoints, the aromatic compounds desaminotyrosine (DAT) and desaminotyrosyl-tyrosine (DATT) were successfully used to functionalize linear and star-shaped oligoethylene glycol (lOEG and sOEG) as previously described. The materials showed properties of surfactants and have potential to be used for solubilization of lipophilic drugs in water. Furthermore, the materials are susceptible for H2O2 degradation as determined by MALDI-ToF MS analyses. This is important for potential in vivo applications, as macrophages can release reactive oxygen species (ROS) under inflammatory conditions. As it is known that surfactant solutions of high concentration can lead to cell lysis, the effects of OEG-DAT(T) solutions on murine RAW macrophages were investigated. Even at highest OEG-DAT(T) concentration of 1000 µg·mL-1 the viability of the RAW cells was not significantly impaired. Additionally, the polymers were incubated with whole human blood and the production of inflammatory cytokines such as the tumor necrosis factor (TNF)-α and interleukin (IL)-6 was determined. Only at high concentrations, the OEG-DAT(T) solution induced low levels of TNF-α and IL-6, indicating that a mild inflammatory reaction could be expected when such high OEG-DAT(T) concentrations are applied in vivo. Similarly, the OEG-DAT(T) solution did not induce ROS in monocytes and neutrophils after incubation with whole human blood. Conclusively, the data presented here demonstrate that OEG-DAT(T) do not lead to a substantial activation of the innate immune mechanisms and could therefore be investigated for solubilizing pharmaceutical agents.

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Production of viable piglets for the first time using sperm derived from ectopic testicular xenografts

Reproduction ◽

10.1530/rep-09-0509 ◽

2010 ◽

Vol 139 (2) ◽

pp. 331-335 ◽

Cited By ~ 52

Author(s):

Michiko Nakai ◽

Hiroyuki Kaneko ◽

Tamas Somfai ◽

Naoki Maedomari ◽

Manabu Ozawa ◽

...

Keyword(s):

Testicular Tissue ◽

Blastocyst Stage ◽

Large Mammals ◽

Immunodeficient Mice ◽

Neonatal Piglets ◽

Porcine Oocyte ◽

Large Animals ◽

First Time

Xenografting of testicular tissue into immunodeficient mice is known to be a valuable tool for facilitating the development of immature germ cells present in mammalian gonads. Spermatogenesis in xenografts and/or in vitro embryonic development to the blastocyst stage after ICSI of xenogeneic sperm has already been reported in large animals, including pigs; however, development of the embryos to term has not yet been confirmed. Therefore, in pigs, we evaluated the in vivo developmental ability of oocytes injected after ICSI of xenogeneic sperm. Testicular tissues prepared from neonatal piglets, which contain seminiferous cords consisting of only gonocytes/spermatogonia, were transplanted under the back skin of castrated nude mice. Between 133 and 280 days after xenografting, morphologically normal sperm were recovered, and a single spermatozoon was then injected into an in vitro matured porcine oocyte. After ICSI, the oocytes were electrostimulated and transferred into estrus-synchronized recipients. Two out of 23 recipient gilts gave birth to six piglets. Here, we describe for the first time that oocytes fertilized with a sperm from ectopic xenografts have the ability to develop to viable offspring in large mammals.

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Efficacy of Synthetic Peptides RP-1 and AA-RP-1 against Leishmania SpeciesIn VitroandIn Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05349-11 ◽

2011 ◽

Vol 56 (2) ◽

pp. 658-665 ◽

Cited By ~ 14

Author(s):

Marie Crisel B. Erfe ◽

Consuelo V. David ◽

Cher Huang ◽

Victoria Lu ◽

Ana Claudia Maretti-Mira ◽

...

Keyword(s):

Intravenous Administration ◽

Synthetic Peptides ◽

Antileishmanial Activity ◽

Host Defense Peptides ◽

Content Type ◽

Causative Agents ◽

Antileishmanial Activities ◽

Conventional Antibiotics

ABSTRACTHost defense peptides are naturally occurring molecules that play essential roles in innate immunity to infection. Based on prior structure-function knowledge, we tested two synthetic peptides (RP-1 and AA-RP-1) modeled on the conserved, microbicidal α-helical domain of mammalian CXCL4 platelet kinocidins. These peptides were evaluated for efficacy againstLeishmaniaspecies, the causative agents of the group of diseases known as leishmaniasis.In vitroantileishmanial activity was assessed against three distinctLeishmaniastrains by measuring proliferation, metabolic activity and parasite viability after exposure to various concentrations of peptides. We demonstrate that micromolar concentrations of RP-1 and AA-RP-1 caused dose-dependent growth inhibition ofLeishmaniapromastigotes. This antileishmanial activity correlated with rapid membrane disruption, as well as with a loss of mitochondrial transmembrane potential. In addition, RP-1 and AA-RP-1 demonstrated distinct and significantin vivoantileishmanial activities in a mouse model of experimental visceral leishmaniasis after intravenous administration. These results establish efficacy of RP-1 lineage synthetic peptides againstLeishmaniaspeciesin vitroand after intravenous administrationin vivoand provide further validation of proof of concept for the development of these and related systemic anti-infective peptides targeting pathogens that are resistant to conventional antibiotics.

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