Hamster Kidney Cells Research Articles

Substrate Optimization in Baby Hamster Kidney Cell Culture for Foot and Mouth Disease Virus Vaccine Using the Taguchi Method.

Cell culture is one of the most commonly used techniques in the production of biological products. Many physical and chemical parameters may affect cell growth and proliferation. This study was conducted to investigate the effect of chemical components as supplements using the experimental design method, which aimed at reducing the number of experiments. For this purpose, supplements including chemical components using four levels, with three replications in suspension and batch culture conditions, were examined for 72 hours using the Taguchi experimental design method. From the experiments, it was concluded that the culture media composition had a significant impact on final cell count and pH. High concentrations of different media composition alone were insufficient to ensure higher cell count. According to the results, this insufficiency was associated with an increase of 20% in the number of final cells. In the majority of cultures, the number of final cells at 48 hours increased relative to the number of final cells at 24 hours after culturing the cells.

Flock House Virus RNA1 with a Long Heterologous Sequence at the 3'-end Can Replicate in Mammalian Cells and Mediate Reporter Gene Expression.

Flock House virus (FHV), an insect RNA virus, has a bipartite genome. FHV RNA1 can be packaged in turnip yellow mosaic virus (TYMV) as long as the FHV RNA has a TYMV sequence at the 3'-end. The encapsidated FHV RNA1 has four additional nucleotides at the 5'- end. We investigated whether the recombinant FHV RNA1 could replicate in mammalian cells. To address this issue, we prepared in vitro transcribed FHV RNAs that mimicked the recombinant FHV RNA1, and introduced them into baby hamster kidney (BHK) cells. The result showed that the recombinant FHV RNA1 was capable of replication. An eGFP gene inserted into the frame with B2 gene of the FHV RNA1 was also successfully expressed. We also observed that eGFP expression at the protein level was strong at 28°C but weak at 30°C. Sequence analysis showed that the 3'-ends of the RNA1 and RNA3 replication products were identical to those of the authentic FHV RNAs. This indicates that FHV replicase correctly recognized an internally-located replication signal. In contrast, the 5'-ends of recombinant FHV RNA1 frequently had deletions, indicating random initiation of (+)-strand synthesis.

Probiotic Lactobacillus and Bifidobacterium strains possess safety characteristics, antiviral activities and host adherence factors revealed by genome mining.

Probiotics belonging to Lactobacillus and Bifidobacterium spp. have been exploited for their health benefits in treatment and prevention of many pathological conditions and promoting human health. Recent advances in understanding probiotics-human interaction through microbiome research in the context of various medical conditions suggest their provisional role in preventive, personalized, and predictive medicine. To streamline their application in disease prevention, development of personalized-based treatments, or their use as biomarkers for predictive diagnosis, in vitro screening for strains with potential probiotic properties should be performed. In this work, we aimed to emphasize the probiotic features of four Lactobacillus and two Bifidobacterium probiotic strains which showed antagonistic properties against microbial pathogens. Firstly, cytotoxicity assessment of cell-free preparations from these strains was performed using a baby hamster kidney (BHK) cells and cell viability was measured by means of sulfo-rhodamine B stain. Secondly, Newcastle disease (ND) and infectious bursal disease (IBD) viruses which pose a great threat in infected poultry were used for assessing antiviral activity of probiotics. Thirdly, the genomes of six probiotic strains were used to identify genes encoding host adherence factors that mediate interaction with human tissues. Probiotic preparations exhibited insignificant toxicity as indicated by the high survival rate of BHK cells (surviving fraction varied from 0.82 to 0.99) as compared to the untreated control. Cell-free preparations of probiotics mixed with equal volume of ND and IBD viruses (106 and 104 Tissue Culture Infectious Dose 50, respectively) reduced the titer of ND and IBD viruses on chicken embryo fibroblast cells. Genome mining analysis revealed that the draft genomes of these strains were predicted to encode LPXTG-containing proteins, surface layer proteins, tight adherence pili, sortase-dependent pili, fibronectin, or collagen binding proteins and other factors that adhere to human tissues such as mucus. Such adherence factors enable probiotic bacteria to interact and colonize the host. Taken together, safety privileges, antiviral activities, and genomically encoded host interaction factors confirmed probiotic features of the six probiotic strains and their potential in promoting human health.

A novel method to rescue and culture duck Astrovirus type 1 in vitro

BackgroundReverse genetics systems enable the manipulation of viral genomes and therefore serve as robust reverse genetic tools to study RNA viruses. A DNA-launched rescue system initiates the transcription of viral genomic cDNA from eukaryotic promoter in transfected cells, generating homogenous RNA transcripts in vitro and thus enhancing virus rescue efficiency. As one of the hazardous pathogens to ducklings, the current knowledge of the pathogenesis of duck astrovirus type 1 (DAstV-1) is limited. The construction of a DNA-launched rescue system can help to accelerate the study of the virus pathogenesis. However, there is no report of such a system for DAstV-1.MethodsIn this study, a DNA-launched infectious clone of DAstV-1 was constructed from a cDNA plasmid, which contains a viral cDNA sequence flanked by hammerhead ribozyme (HamRz) and a hepatitis delta virus ribozyme (HdvRz) sequence at both terminals of the viral genome. A silent nucleotide mutation creating a Bgl II site in the ORF2 gene was made to distinguish the rescued virus (rDAstV-1) from the parental virus (pDAstV-1). Immunofluorescence assay (IFA) and western blot were conducted for rescued virus identification in duck embryo fibroblast (DEF) cells pre-treated with trypsin. The growth characteristics of rDAstV-1 and pDAstV-1 in DEF cells and the tissue tropism in 2-day-old ducklings of rDAstV-1 and pDAstV-1 were determined.ResultsThe infectious DAstV-1 was successfully rescued from baby hamster kidney (BHK-21) cells and could propagate in DEF cells pre-treated with 1 μg/ml trypsin. Upon infection of DEF cells pre-treated with trypsin, DAstV-1 mRNA copies were identified after serial passaging, and the result showed that rDAstV-1 and pDAstV-1 shared similar replication kinetics. Animal experiment showed that the rDAstV-1 had an extensive tissue tropism, and the virus was capable of invading both the central and the peripheral immune organs in infected ducklings.ConclusionsAn improved DNA-launched reverse genetics system for DAstV-1 was firstly constructed. Infectious virus recovered from BHK-21 cells could propagate in DEF cells pre-treated with trypsin. This is the first report of the successful in vitro cultivation of DAstV-1. We believe this valuable experimental system will contribute to the further study of DAstV-1 genome function and pathogenesis.

Nanoceria: A rare-earth nanoparticle as a promising anti-cancer therapeutic agent in colon cancer

The bioactivity and physical characteristics of CeO2 nanoparticles are significantly dependent on the type of the particle, particle size and defects. Herein, a facile and cost effective microwave-assisted combustion route was used to prepare pure CeO2 nanoparticles (NPs). The samples were thoroughly characterized by various techniques and tested as anti-cancer therapeutic agent in colon cancer. The structural investigations (X-ray Powder Diffraction) revealed the formation of single phase cubic fluorite-type structure of CeO2 with Fm-3m space group. Morphological analysis (FESEM), demonstrated the aggregation of irregular nanoparticles with grain size ~ 20–40 nm. To have more knowledge on the sample morphology, TEM and HRTEM images were taken. TEM image showed ceria NPs with squared shape and particle size in 50–95 nm range. Furthermore, HR-TEM micrographs confirmed the crystallinity of the CeO2 sample, which was explored by calculating the distance between the fringes in the corresponding Fourier transformed images. The BET specific surface area (SSA) of as-synthesized ceria nanoparticles, was observed to be 25 m2/g. UV–vis spectral analysis reveals that the direct band gap of CeO2 nanoparticles is about 2.8 eV.It was interesting to observe that, the as prepared NPs exhibit remarkable differential cytotoxicity towards healthy (BHK121, Baby Hamster Kidney cells) and human Colon cancer cells (HCT116). Nuclear fragmentation assay indicated clear DNA fragmentation in HCT116 cells, whereas intact nucleus was observed in normal cells, suggesting ceria induced apoptotic cell death in colon cancer cells. In sum, MACS generated ceria nanoparticles could uncover a new avenue in cancer therapy.

Sec62 Suppresses Foot-and-Mouth Disease Virus Proliferation by Promotion of IRE1α-RIG-I Antiviral Signaling.

Foot-and-mouth disease virus (FMDV) is highly infectious and causes a major plague in animal farming. Unfolded protein response is one of the major cellular responses to pathogenic infections, which performs a crucial role in cell survival, apoptosis, and antiviral innate immune response. In this study, we showed that FMDV infection activated two unfolded protein response branches (PERK-eIF2α and ATF6 signaling) in both baby hamster kidney cells (BHK-21) and porcine kidney (PK-15) cells, whereas it suppressed the IRE1α-XBP1 signaling by decreasing IRE1α level. Further study revealed IRE1α signaling as an important antiviral innate immune mechanism against FMDV. Sec62, the transport protein, was greatly decreased at the late stages of FMDV infection. By overexpression and knockdown study, we also found that the expression of Sec62 was positively involved in the levels of IRE1α and RIG-I and subsequent activation of downstream antiviral signaling pathways in FMDV-infected PK-15 cells. Taken together, our study demonstrates that Sec62 is an important antiviral factor that upregulates IRE1α-RIG-I-dependent antiviral innate immune responses, and FMDV evades antiviral host defense mechanism by downregulating Sec62-IRE1α/RIG-I.

Contribution of Sodium-Calcium Exchanger Isoform-3 in Aβ1-42 Induced Cell Death

Aβ1-42 is a peptide which can alter intracellular calcium (Ca2+) homeostasis and subsequently cause cell death. The molecular mechanisms underlying Aβ1-42 induced Ca2+ dyshomeostasis and neurodegeneration are not fully elucidated. The third isoform of the sodium-calcium exchanger (NCX) family, NCX3, plays a vital role in adjusting excitable cells Ca2+ homeostasis. In this study, using NCX3 stably transfected baby hamster kidney (BHK) cells, we aim to study the role of NCX3 against Aβ1-42 induced cell death. Our result demonstrates that NCX3 stably transfected cells (BHK-NCX3) are more resistant to Aβ1-42 aggregation compared to their wild types (BHK-WT). The increased cellular loss in BHK-WT was correlated to increased cytoplasmic Ca2+ concentration ([Ca2+]i) induced by Aβ1-42. These results present that NCX3 protects against Aβ1-42 induced cell death and proclaim NCX3 and its downstream molecular pathways as critical components that take part in Aβ1-42 neurotoxicity. Our study substantiates molecular events through which Aβ1-42 can induce neurodegeneration such as Alzheimers disease and marks potential targets to slow down neuropathogenesis.

Detection of Cell Viability via Fluorescence Labeling of Silicate Phosphor with a Low-Temperature Superlong Persistent Luminescence

Cell viability detection is usually accompanied by large errors and toxicity. For the accuracy and safety of detection, it is urgent to develop a practical approach to realize the real-time monitoring of cell viability. Here, a low-temperature persistent phosphor was employed to facilitate the detection of cell viability. Stable low-temperature persistent luminescence originating from Ba4Si6O16:Eu2+, Pr3+ (BSEP) was developed, which could be quantified to describe the viability of baby Syrian hamster kidney (BHK-21) cells. The defect state structure guarantees the excellent persistent luminescence at 193 K of more than 200 h of the as-prepared BSEP phosphor, which ensures the long-term biological application. The structural thermal stability and water tolerance of BSEP suggest the feasibility of the practical application. Our results provide the underlying insights of the low-temperature persistent phosphors for real-time biological detection.

Dissecting the Components of Sindbis Virus from Arthropod and Vertebrate Hosts: Implications for Infectivity Differences

Sindbis virus (SINV) is an enveloped, single-stranded RNA virus, which is transmitted via mosquitos to a wide range of vertebrate hosts. SINV produced by vertebrate, baby hamster kidney (BHK) cells is more than an order of magnitude less infectious than SINV produced from mosquito (C6/36) cells. The cause of this difference is poorly understood. In this study, charge detection mass spectrometry was used to determine the masses of intact SINV particles isolated from BHK and C6/36 cells. The measured masses are substantially different: 52.88 MDa for BHK derived SINV and 50.69 MDa for C6/36 derived. Further analysis using several mass spectrometry-based methods and biophysical approaches indicates that BHK derived SINV has a substantially higher mass than C6/36 derived because in the lipid bilayer, there is a higher portion of lipids containing long chain fatty acids. The difference in lipid composition could influence the organization of the lipid bilayer. As a result, multiple stages of the viral lifecycle may be affected including assembly and budding, particle stability during transmission, and fusion events, all of which could contribute to the differences in infectivity.

EVALUATION OF SUITABILITY OF METHODS FOR TESTING BLOOD PROTEIN HYDROLYSATES FOR THEIR BIOLOGICAL ACTIVITY AND TOXICITY

The use of high quality nutrient media comprising protein hydrolysates as the main components for maintaining their nutrient value is a critical prerequisite for appropriate fnal product preparation in FMD vaccine biotechnology. The incoming control of raw materials, in particular testing blood protein hydrolysates of different batches for their quality, is of considerable importance. Currently, there are different methods for the determination of hydrolysate efcacy using cell cultures and unicellular organisms as test systems. We used suspended baby hamster kidney (ВНК-21) cell line as a model for testing blood protein hydrolysates in the raw materials intended for FMD vaccine production for their biological activity and toxicity. The validation of the proposed method was carried out to confrm the reliability of the test results obtained. The following main validation characteristics were determined through testing 560 blood protein hydrolysate samples: trueness, specifcity, sensitivity, as well as precision under repeatability and reproducibility conditions. The validation results for the proposed method demonstrated high reliability (Fisher’s criterion – 0.021; ≤0.050) and full compliance with acceptance criteria.

MAPK Signaling Influenced by Oxygen Environments

Mammalian cells thrive in a variety of different environments. In tissue culture there are many variables cells are exposed to; the impact of oxygen gas is one that is not well understood in relation to signaling pathways. In tissue culture, cells are commonly grown in atmospheric oxygen (~21%), yet the environment of many cells in vivo is considerably less (1% – 14% depending on the cells type). The increased oxygen content can cause higher levels of reactive oxide species (ROS). When ROS overwhelm the cell, normal cellular responses are affected. Extracellular kinase 1 and 2 (ERK 1/2) are proteins know to be directly modified by ROS, sometimes resulting in kinase inhibition. In this study we examined responses of the MAPK pathway to epidermal growth factor (EGF) in normoxic (6% O2) and atmospheric (21% O2) conditions. Baby hamster kidney (BHK‐21) or Human Dermal Epithelial (HMEC‐1) cells were grown and maintained in atmospheric conditions. The cells were then seeded and either kept under atmospheric conditions (normal tissue culture conditions) or moved to 6% oxygen in a Bactrox™(with all other conditions remaining constant). These cells were grown for two days until approximately 80–90% confluent and then they were serum starved and treated with EGF (0–30 minutes) in the two environments. They were harvested on ice in their respective environments. The lysates were then removed and frozen. After lysis the two conditions were treated in atmospheric oxygen. We hypothesized that the ERK 1/2 pathway responses would be different under the conditions of 6% versus 21% oxygen. ERK 1/2 phosphorylation status was assessed in parallel with kinase activity. Also explored was the difference in cell growth between the two environments. Our results show subtle differences in the peaks of phosphorylation and in the threshold of responses as well as differences in activity of the harvested ERK. Our work will lead to an enhanced understanding of how cellular signaling pathways are impacted by oxygen levels.Support or Funding InformationNIH grant # R15 GM110634‐01A1 to C.A.C., and Kennesaw State University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Simultaneous Delivery of CRISPR/Cas and Donor DNA using Cell‐Penetrating Peptide‐Adaptors

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are utilized as a form of adaptive immunity in prokaryotes. CRISPR detects and eliminates foreign nucleic acids such as those found in bacteriophage. To achieve this form of ‘memory of infection,’ CRISPR associated proteins (Cas) form complexes with RNA to cause double-stranded breaks (DSBs) in the invader's sequence. The invader's DNA between these breaks is incorporated within the CRISPR locus in the bacterial genome. CRISPR/Cas9 has been a breakthrough for genome editing due to its specificity and potential to allow genome editing in patients, where it could be used to treat many diseases such as cancer or hemophilia. The present work aims to enhance CRISPR efficiency by delivery of Cas9 complexed with guide RNA (crRNA and tracRNA) to the nuclei of living cells using a novel cell-penetrating peptide (CPP)-adaptor system. CPPs have long been used to deliver biomolecules to living cells, but CPP-cargos remained trapped in endosomes. To solve this problem, we used a novel CPP adaptor, TAT-CaM, a fusion protein in which the CPP from HIV TAT is fused to human calmodulin (CaM). When Ca2+ is present, TAT-CaM binds to an engineered calmodulin binding site (CBS) on cargo proteins (e.g. CBS-Cas9). After endocytosis, TAT-CaM dissociates from cargos as Ca2+ concentrations drop during trafficking. Like other CPPs, TAT-CaM remains trapped in endosomes but cargos efficiently escape. In the present study, biolayer interferometry (BLI) and confocal microscopy were used to demonstrate Ca2+-dependent binding and delivery of CRISPR complexes to the nuclei of cells. We have employed a similar scheme to deliver in parallel donor DNA that can be used by the cell's homologous recombination machinery to effect editing. Donor DNA is internalized simultaneously with CRISPR/Cas using a CBS-tamavidin fusion protein that binds both TAT-CaM and biotinylated donor DNA. Preliminary results show that CBS-tamavidin is wellexpressed in E. coli and readily purified using immobilized metal affinity chromatography. BLI analysis showed that CBS-tamavidin binds TAT-CaM affinity in the presence of Ca2+ while simultaneously binding biotinylated DNA, both with high affinity (fast on/slow off). Confocal microscopy showed that CBS-Cas9 and CBS-tamavidin/biotin-DNA enter cells in the presence TAT-CaM but not in the absence. Support or Funding Information National Institutes of Health grant GM R15120691 Preliminary results for delivery of CBS-Cas9 in Baby Hamster Kidney(BHK) cells using confocal microscopy. CRISPR RNAs are labeled with Alexa 550 red, the nucleus is labeled with NucBlue, the Cas9 and TAT CaM are unlabeled. The first column only shows the CRISPR RNA, the second column only shows the nucleus and the third column shows the composite of the first and the second column. The first row and the second row show the cells with CRISPR complex in 10 and 20 minutes respectively. BioLayer Interferometry (BLI) analysis of CBS-Cas9. (A) Association and dissociation of CBS-Cas9 (analyte at concentrations starting from 200nM to 50nM) in TAT CaM(ligand at 1uM) and a calcium-containing buffer respectively. (B) CBS-Cas9 dissociation in EDTA(chelates calcium) from 200nM to 50nM. (C) Kinetic parameters for CBS-Cas9 from A and B. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rapid and Sparse Labeling of Neurons Based on the Mutant Virus-Like Particle of Semliki Forest Virus.

Sparse labeling of neurons contributes to uncovering their morphology, and rapid expression of a fluorescent protein reduces the experiment range. To achieve the goal of rapid and sparse labeling of neurons in vivo, we established a rapid method for depicting the fine structure of neurons at 24 h post-infection based on a mutant virus-like particle of Semliki Forest virus. Approximately 0.014 fluorescent focus-forming units of the mutant virus-like particle transferred enhanced green fluorescent protein into neurons in vivo, and its affinity for neurons in vivo was stronger than for neurons in vitro and BHK21 (baby hamster kidney) cells. Collectively, the mutant virus-like particle provides a robust and convenient way to reveal the fine structure of neurons and is expected to be a helper virus for combining with other tools to determine their connectivity. Our work adds a new tool to the approaches for rapid and sparse labeling of neurons in vivo.

CX3CR1-CX3CL1-dependent cell-to-cell Japanese encephalitis virus transmission by human microglial cells

The neurotropic Japanese encephalitis virus (JEV) is responsible for Japanese encephalitis, an uncontrolled inflammatory disease of the central nervous system. Microglia cells are the unique innate immune cell type populating the brain that cross-communicate with neurons via the CX3CR1-CX3CL1 axis. However, microglia may serve as a viral reservoir for JEV. Human microglia are able to transmit JEV infectivity to neighbouring cells in a cell-to-cell contact-dependent manner. Using JEV-treated human blood monocyte-derived microglia, the present study investigates molecular mechanisms behind cell-to-cell virus transmission by human microglia. For that purpose, JEV-associated microglia were co-cultured with JEV susceptible baby hamster kidney cells under various conditions. Here, we show that microglia hosting JEV for up to 10 days were able to transmit the virus to susceptible cells. Interestingly, neutralizing anti-JEV antibodies did not completely abrogate cell-to-cell virus transmission. Hence, intracellular viral RNA could be a contributing source of infectious virus material upon intercellular interactions. Importantly, the CX3CL1-CX3CR1 axis was a key regulator of cell-to-cell virus transmission from JEV-hosting human microglia. Our findings suggest that human microglia may be a source of infection for neuronal populations and sustain JEV brain pathogenesis in long-term infection. Moreover, the present work emphasizes on the critical role of the CX3CR1-CX3CL1 axis in JEV pathogenesis mediating transmission of infectious genomic JEV RNA.

ABCA1 Agonist Mimetic Peptide CS-6253 Induces Microparticles Release From Different Cell Types by ABCA1-Efflux–Dependent Mechanism

BackgroundSmall peptides based on the C-terminal domain of apo E have recently been proposed as ATP-binding cassette transporter A1 (ABCA1) agonist with therapeutic potential. Previous work has shown that a novel synthetic peptide, CS-6253, acts synergistically with apolipoprotein A-I or alone to generate high-density lipoprotein (HDL) particles; we have also shown that cells can release microparticles (50-350 nm in apparent diameter) in an ABCA1- and apolipoprotein A-I-dependent manner. The purpose of this study was to explore the ability of a novel synthetic peptide CS-6253 to induce microparticle release from various cell lines in the process of HDL biogenesis. MethodsThe effects of CS-6253 on microparticle formation through the ABCA1 transporter were examined in vitro using cell-based systems and pharmacologic manipulations. ResultsIn cell-based systems combined with fast performance liquid chromatography and nano-sight-tracking analysis, we show that ABCA1 and CS-6253 mediate and increase the production of microparticles containing cholesterol. CS-6253 in baby hamster kidney cells not expressing ABCA1 (baby hamster kidney mock cells) did not alter cholesterol removal across the plasma membrane in the absence of ABCA1 expression even at high concentrations. We report that CS-6253 is not cytotoxic. ConclusionsThe present study shows that CS-6253 generates cholesterol containing microparticles with size heterogeneity (100-350 nm) in an ABCA1-dependent manner. We show that microparticles contribute to cell cholesterol efflux from monocyte-macrophage cells. At high doses, CS-6253 is not able to extract cholesterol from cells not expressing ABCA1, indicating that CS-6253 requires ABCA1 cooperation for cholesterol mobilization. We conclude that CS-6253 is an ABCA1 agonist peptide that promotes cellular cholesterol efflux through HDL biogenesis and microparticle formation.

Hydrogel Formation by an Aromatic Analogue of a β-Amyloid Fragment, Aβ16–22: A Scaffold for 3D Cell Culture

A short 7-residue stretch from β-amyloid, Aβ16–22 (Ac-KLVFFAE-am), yields typical amyloid-like fibrils at neutral pH. The Phe–Phe cassette present in the peptide is believed to be critical for its self-assembly. We report that the aromatic analogue Aβ16–22(F20Y) forms self-supporting soft gels at concentrations ≥2 mM even though the end-capped parent peptide does not form hydrogel up to 20 mM (1.8% w/w) concentration. The hydrogel is made up of distinct amyloid-like fibers. The storage modulus of 20 mM gel is ∼3–5-fold higher than the loss modulus in the 2–3000 rad/s angular frequency range, indicating distinct elastic properties. The hydrogel supports the growth of rat pancreatic cells (RIN-5F), human embryonic kidney cells (HEK-293), baby hamster kidney cells (BHK-21), and human neuroblastoma cells (IMR-32). The cells grow in clusters as is anticipated in a three-dimensional matrix. The rat pancreatic cells produced insulin, suggesting that they are functional inside the gel.

Synthesis, characterization and cytotoxicity of polyethylene glycol-encapsulated CdTe quantum dots

Semiconductor quantum dots (QDs) offer tremendous scope to be used in the field of energy, imaging, sensors and optoelectronic devices. However, several issues regarding their hydrophilicity, stability and cytotoxicity remain unresolved. One of the methods to render CdTe QDs water-soluble is to functionalize the surface with carboxylate groups by the use of heterobifunctional ligands such as 3-mercaptopropionic acid (MPA). Following this motif, we report the synthesis of CdTe QDs using the non-aqueous organometallic route, ligand exchange with MPA was performed to replace the initial surface passivation ligands trioctylphosphine oxide (TOPO) and hexadecylamine, and finally, the prepared MPA-CdTe QDs were encapsulated into the biocompatible polyethylene glycol. Our results show that pegylation of CdTe QDs provided minimal cytotoxicity against baby hamster kidney (BHK 21) cells even at high levels. The IC50 values of pegylated CdTe QDs were higher than the non-pegylated ones with a % viability ranges from 89:90% at a concentration range of 0.01:0.03 nM. Currently, available ligand exchange procedures lead to significant loss of quantum yields. In our study, the photoluminescence (PL) emission of the MPA capped CdTe QDs decreased gradually as the pH increased from 4 to 10 while after pegylation the prepared QDs exhibited significantly enhanced PL efficiency and storage stability.

Characterization of liver specific promoters in a foamy viral vector pMD09.

Foamy viruses (FVs) or spumaviruses are retroviruses that are explored as vectors for gene therapy. The good feature of foamy viruses is its broad tropism; however, their infections result in non-targeted gene expression. Here, we attempted to design the liver targeted viral gene delivery by employing liver specific gene promoters like albumin (ALB), transthyretin (TTR) and hepatitis B virus (HBV) promoters. We compared the relative gene expression of liver specific promoters versus the U3 promoter in liver cell line (HepG2) and non-liver cell lines: human fibrosarcoma cell line (HT1080), baby hamster kidney cell line (BHK), human embryonic kidney cell line (HEK 293T) and cervical cancer cell line (HeLa). We have found that the promoter exchange didn't affect viral assembly. The ability to drive gene expression was best with TTR promoter which was followed by HBV and ALB promoter. The use of TTR, HBV and ALB promoters are helpful in achieving liver specific gene expression. Keywords: foamy virus; gene therapy; liver; albumin; transthyretin promoter; HBV promoter.

Production and Characterisation of Acellular Human Liver Matrix: Potential Auxiliary Liver Graft

Shortage of donor organs combined with ever increasing liver disease has directed attention towards growing implantable liver tissue in the laboratory setting. Recent developments in decellularisation and recellularisation techniques have opened exciting facets for liver whole organ reengineering. The aim of this study was to develop a decellularisation protocol for human liver, assess the structural and biochemical integrity and assess the biocompatibility of the acellular matrix. Discarded human livers were split and the left lateral and right posterior lobes with a mean weight of 399 ± 162 g were used (n=8 lobes). Perfusion decellularisation was carried out through the hepatic artery and portal vein. Hypotonic buffer, 0.1 % (w/v) sodium dodecyl sulphate, hypertonic buffer and nuclease solutions were used to remove cells plus nuclear material. Following several iterations of a previously patented Leeds University decellularisation protocol, greater than 91.0 % (w/w) DNA was removed from all areas of liver tissue. Fluoroscopy confirmed integrity of vascular tree. Histological analysis demonstrated lack of cells and maintenance of the portal triad histioarchitecture. Immunohistochemistry demonstrated positive staining for key extracellular proteins such as collagen type I and III, fibronectin and laminin. Scanning electron microscopy showed an intact extracellular matrix. Biochemical assessment confirmed significant reduction in glycosaminoglycan content, but there was increase in collagen content, the latter likely due to extraction by dry weight of other soluble components in the matrix. The acellular tissues and extracts were not cytotoxic to either murine 3T3 or baby hamster kidney cells. Preliminary recellularisation (n=3 lobes) showed the matrix to be biocompatible to primary cryopreserved human hepatocytes seeded at 30,000 cells. The study has developed a suitable protocol for decellularisation of human liver lobes, without adversely affecting the extracellular matrix. The biocompatible acellular scaffold has the potential to be recellularised with hepatocytes, with a view to engineering a transplantable auxiliary liver graft.

Are the Statins promising antifungal agents against invasive candidiasis?

The medical importance of intra-abdominal candidiasis (IAC) contrasts with the limited number of pharmacological treatment options available and the increasing rate of resistance to antifungal drugs. Thus, the repositioning of compounds in clinical use can contribute to the broadening of treatment possibilities for this infection. Statins, a class of drugs used to reduce cardiovascular event risk, have shown antiparasitic, antibacterial, and antiviral activities; however, their antifungal effects remain poorly studied. In this context, the present study aimed to elucidate the antifungal potential of six statins in vitro, as well as to evaluate the therapeutic use of fluvastatin in a mouse model of IAC. The biological effects of statins were evaluated against Candida spp., through the determination of the minimum inhibitory concentration (MIC). For the statins that showed activity, the fungicidal concentration, toxicity/selectivity, synergism with azoles and polyenes, phenotypic effects, and activity against virulence factors were also determined. Atorvastatin, rosuvastatin and fluvastatin were highly active, especially against C. albicans (MIC < 1–128 μg.mL−1) and C. glabrata (MIC 32–64 μg.mL−1). Fluvastatin and atorvastatin were selective for C. albicans in baby hamster kidney (BHK) cells. Moreover, all active statins in the antifungal assay showed high selectivity for fungal cells over bacteria. The combination of atorvastatin, rosuvastatin, and fluvastatin with azoles was associated with a synergistic effect. Active statins do not act on the fungal membrane or wall, but instead stimulate farnesol-dependent pathogenicity factors such as yeast-to-hyphal transition and biofilm generation. Fluvastatin treatment was evaluated in a mouse model of IAC, showing stimulation of the extra-hepatic dissemination of C. albicans but improvements in renal, splenic, and hepatic histological aspects. In conclusion, statins have potent antifungal activity in vitro, but the therapeutic effect in vivo is restricted to their anti-inflammatory activity.