Advances in Computational and Bioinformatics Tools and Databases for Designing and Developing a Multi-Epitope-Based Peptide Vaccine.

A vaccine is defined as a biologic preparation that trains the immune system, boosts immunity, and protects against a deadly microbial infection. They have been used for centuries to combat a variety of contagious illnesses by means of subsiding the disease burden as well as eradicating the disease. Since infectious disease pandemics are a recurring global threat, vaccination has emerged as one of the most promising tools to save millions of lives and reduce infection rates. The World Health Organization reports that immunization protects three million individuals annually. Currently, multi-epitope-based peptide vaccines are a unique concept in vaccine formulation. Epitope-based peptide vaccines utilize small fragments of proteins or peptides (parts of the pathogen), called epitopes, that trigger an adequate immune response against a particular pathogen. However, conventional vaccine designing and development techniques are too cumbersome, expensive, and time-consuming. With the recent advancement in bioinformatics, immunoinformatics, and vaccinomics discipline, vaccine science has entered a new era accompanying a modern, impressive, and more realistic paradigm in designing and developing next-generation strong immunogens. In silico designing and developing a safe and novel vaccine construct involves knowledge of reverse vaccinology, various vaccine databases, and high throughput techniques. The computational tools and techniques directly associated with vaccine research are extremely effective, economical, precise, robust, and safe for human use. Many vaccine candidates have entered clinical trials instantly and are available prior to schedule. In light of this, the present article provides researchers with up-to-date information on various approaches, protocols, and databases regarding the computational designing and development of potent multi-epitope-based peptide vaccines that can assist researchers in tailoring vaccines more rapidly and cost-effectively.

Investigation of Antimicrobial Activity and Biocompatibility of Biogenic Silver Nanoparticles Synthesized using Syzigyum cymosum Extract.

Nanotherapeutics has emerged as the most sought after approach to tackle the menace of drug-resistant pathogenic bacteria. Among others, biogenic silver nanoparticles (bAgNPs) synthesized using medicinal plant extracts demonstrate promising antibacterial propensity with excellent biocompatibility. Herein, bAgNPs were synthesized through the green chemistry approach using Syzygium cymosum leaf extract as a reducing agent at different pH values (i.e., 5, 7, 8, and 10). The average size of bAgNPs synthesized at pH 5, 7, 8, and 10 was 23.3, 21.3, 17.2, and 35.3 nm, respectively, and all the nanoparticles were negatively charged. Their antibacterial potential was investigated against Bacillus subtilis, Escherichia coli DH5α, E. coli K12, enteropathogenic E. coli, and Salmonella typhi. The highest antibacterial activity was exhibited by bAgNPs synthesized at pH 8 against all the tested bacterial strains, which can be attributed to their small size and greater surface area to volume ratio. The bAgNPs demonstrated the highest zone of inhibition (29.5 ± 0.8 mm) against B. subtilis through oxidation of membrane fatty acids that resulted in the formation of the malondialdehyde-thiobarbituric acid (MDA-TBA) adduct. However, bAgNPs demonstrated excellent hemocompatibility with rat and human red blood cells. Biogenic AgNPs synthesized at pH 8 also exhibited biocompatibility in terms of liver and kidney function biomarkers. Furthermore, hematoxylin and eosin staining of the tissue sections of vital organs (i.e., liver, kidneys, lungs, heart, spleen, and brain) also confirmed the biocompatibility of bAgNPs.

Modulation of Local Cellular Activities using a Photothermal Dye-Based Subcellular-Sized Heat Spot.

Thermal engineering at the microscale, such as the regulation and precise evaluation of the temperature within cellular environments, is a major challenge for basic biological research and biomaterials development. We engineered a polymeric nanoparticle having a fluorescent temperature sensory dye and a photothermal dye embedded in the polymer matrix, named nanoheater-thermometer (nanoHT). When nanoHT is illuminated with a near-infrared laser at 808 nm, a subcellular-sized heat spot is generated in a live cell. Fluorescence thermometry allows the temperature increment to be read out concurrently at individual heat spots. Within a few seconds of an increase in temperature by approximately 11.4 °C from the base temperature (37 °C), we observed the death of HeLa cells. The cell death was observed to be triggered from the exact local heat spot at the subcellular level under the fluorescence microscope. Furthermore, we demonstrate the application of nanoHT for the induction of muscle contraction in C2C12 myotubes by heat release. We successfully showed heat-induced contraction to occur in a limited area of a single myotube based on the alteration of protein-protein interactions related to the contraction event. These results demonstrate that even a single heat spot provided by a photothermal material can be extremely effective in altering cellular functions.

Functionalized Concave Cube Gold Nanoparticles as Potent Antimicrobial Agents against Pathogenic Bacteria.

Gold (Au) is an inert metal in a bulk state; however, it can be used for the preparation of Au nanoparticles (i.e., AuNPs) for multidimensional applications in the field of nanomedicine and nanobiotechnology. Herein, monodisperse concave cube AuNPs (CCAuNPs) were synthesized and functionalized with a natural antioxidant lipoic acid (LA) and a tripeptide glutathione (GSH) because different crystal facets of AuNPs provide binding sites for distinct ligands. There was an ∼10 nm bathochromic shift of the UV-vis spectrum when CCAuNPs were functionalized with LA, and the size of the as-synthesized monodisperse CCAu nanoparticles was 76 nm. The LA-functionalized CCAu nanoparticles (i.e., CCAuLA) showed the highest antibacterial activity against Bacillus subtilis. Both fluorescence images and scanning electron microscopy images confirm the damage of the bacterial cell wall as the mode of antibacterial activity of CCAuNPs. CCAuNPs also cause the oxidation of bacterial cell membrane fatty acids to produce reactive oxygen species, which pave the way for the death of bacteria. Both CCAu nanoparticles and their functionalized derivatives showed excellent hemocompatibility (i.e., percentage of hemolysis is <5% at 80 µg of AuNPs) to human red blood cells and very high biocompatibility to HeLa, L929, and Chinese hamster ovary-green fluorescent protein (CHO-GFP) cells. Taken together, LA and GSH enhance the antibacterial activity and biocompatibility, respectively, of CCAu nanoparticles that interact with the bacteria through Coulomb as well as hydrophobic interactions before demonstrating antibacterial propensity.

Nanodiamond-chitosan functionalized hernia mesh for biocompatibility and antimicrobial activity.

Polypropylene (PP) mesh is most commonly used for the treatment of hernia and pelvic floor construction. However, some of the patients have a few complications after surgery due to the rejection or infection of the implanted meshes. The poor biocompatibility of PP mesh, low wettability results in poor cell attachment/proliferation and restricts the loading of antibacterial agent, leading to a slow healing process and high risk of infection after surgery. Here in this study, a new technique has been employed to develop a novel antimicrobial and biocompatible PP mesh modified with bioactive chitosan and functionalized nanodiamond (FND) for infection inhibition and acceleration of the healing process. An oxygen plasma treatment PP mesh was used then chitosan was strongly attached to the surface of the PP fibers. Subsequently, FND as an antibacterial agent was loaded into the chitosan modified PP fiber to provide desired antibacterial functions. The meshes were characterised with XRD, FTIR, SEM, EDX, water contact angle, confocal, and optical microscopy. The modified PP mesh with chitosan and FND showed a significant increase in its hydrophilicity and L929 fibroblast cell attachment. Furthermore, the modified mesh exhibited great antibacterial efficiency against Escherichia coli. Therefore, the newly developed technique to modify PP mesh could be a promising technique to generate a biocompatible PP mesh to accelerate the healing process and reduce the risk of infection after surgery.

In Vitro Delivery of Cell Impermeable Phallotoxin Using Cationic Liposomes Composed of Lipids Bearing Lysine Headgroup.

Bioactive peptides, which act as biologically active regulators, often require intracellular delivery systems to access their therapeutic targets in the cytosolic space maintaining their bioactivity. Here, we report on the delivery of a polar cell impermeable bioactive peptide, phalloidin, into living HeLa cells with cationic liposomes prepared from lysine-based lipids. Liposome/Alexa Fluor 594 phalloidin complexes were characterized regarding their size and zeta potential, which were 85 ± 38 nm and +24.5 ± 4.21 mV, respectively. The delivery of Alexa Fluor 594 phalloidin into live HeLa cells with K3C14 liposomes was evaluated using a fluorescence activated cell sorter and confocal laser scanning microscopy. The highest Alexa Fluor 594 phalloidin delivery efficiency was 92% when using 200 µg of the cationic lipid/1 × 105 cells seeded at 37 °C. The cellular uptake mechanism for the cationic liposome/Alexa Fluor 594 phalloidin complexes was investigated using various endocytosis inhibitors. We confirmed the complexes were mainly taken up through caveolae-mediated endocytosis. Incubation with bafilomycin A1, which inhibits the acidification of lysosomes, revealed that Alexa Fluor 594 phalloidin did not pass through the lysosomal pathway. Rather, Alexa Fluor 594 phalloidin was released from early endosomes or caveosomes to the cytosol to exhibit its bioactive effects including the multinucleation of HeLa cells.

Three-dimensional directional nerve guide conduits fabricated by dopamine-functionalized conductive carbon nanofibre-based nanocomposite ink printing.

A potential issue in current nerve guides is that they do not transmit electrical nerve impulses between the distal and proximal end of an injured nerve, i.e. a synapse. Conductivity is a desirable property of an ideal nerve guide that is being considered for peripheral nerve regeneration. Most conductive polymers reported for the fabrication of tissue engineering scaffolds, such as polypyrrole and polyaniline, are non-biodegradable and possess weak mechanical properties, and thus cannot be fabricated into 3D structures. Herein, we have designed a new nanocomposite material composed of dopamine, carbon nanofibers (CNF) and polycaprolactone (PCL) for the fabrication of nerve conduits, which facilitates the growth and migration of neurons toward the targeted end of an injured nerve. This support and navigation of the scaffold leads to better sensory and motor function. The results showed that the mechanical properties of the printed PCL increased by 30% in comparison with the pure PCL film, which is comparable with human nerves. The in vitro cell study of human glioma cells showed that the printed lines provided support for neural cell attachment, migration and differentiation toward the targeted end. In contrast, in the absence of printed lines in the scaffold, the cells attach and grow in random directions, forming a flower shape (cell cluster) on the surface of PCL. Thus, the proposed scaffold is a promising candidate for nerve guide application based on its signal transmission and navigating neurons in a correct pathway towards the targeted end.

Correction: Three-dimensional directional nerve guide conduits fabricated by dopamine-functionalized conductive carbon nanofibre-based nanocomposite ink printing.

[This corrects the article DOI: 10.1039/D0RA06556K.].

Investigation of the Antibacterial Activity and in vivo Cytotoxicity of Biogenic Silver Nanoparticles as Potent Therapeutics.

Biogenic nanoparticles are the smartest weapons to deal with the multidrug-resistant "superbugs" because of their broad-spectrum antibacterial propensity as well as excellent biocompatibility. The aqueous biogenic silver nanoparticles (Aq-bAgNPs) and ethanolic biogenic silver nanoparticles (Et-bAgNPs) were synthesized using aqueous and ethanolic extracts of Andrographis paniculata stem, respectively, as reducing agents. Electron microscopic images confirmed the synthesis of almost spherical shaped biogenic silver nanoparticles (bAgNPs). The zeta potentials of the nanoparticles were negative and were -22 and -26 mV for Aq-bAgNPs and Et-bAgNPs, respectively. The antibacterial activity of bAgNPs was investigated against seven pathogenic (i.e., enteropathogenic Escherichia coli, Salmonella typhi, Staphylococcus aureus, Vibrio cholerae, Enterococcus faecalis, Hafnia alvei, Acinetobacter baumannii) and three nonpathogenic (i.e., E. coli DH5α, E. coli K12, and Bacillus subtilis) bacteria at different time points (i.e., 12, 16, 20, and 24 h) in a dose-dependent manner (i.e., 20, 40, and 60 µg) through broth dilution assay, disk diffusion assay, CellToxTM Green uptake assay, and trypan blue dye exclusion assay. The lowest minimum inhibitory concentration value for both the bAgNPs was 0.125 µg. Et-bAgNPs showed the highest antibacterial activity against S. aureus at 60 µg after 16 h and the diameter of inhibited zone was 28 mm. Lipid peroxidation assay using all the bacterial strains revealed the formation of malondialdehyde-thiobarbituric acid adduct due to the oxidation of cell membrane fatty acids by bAgNPs. The bAgNPs showed excellent hemocompatibility against human as well as rat red blood cells. Furthermore, there was no significant toxicity observed when the levels of rat serum ALT, AST, γ-GT (i.e., liver function biomarkers), and creatinine (i.e., kidney function biomarker) were determined.

Evaluation of plasmid DNA stability against ultrasonic shear stress and its in vitro delivery efficiency using ionic liquid [Bmim][PF6].

The hydrophobic ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate [Bmim][PF6] forms nanostructures with negatively charged plasmid DNA through electrostatic interactions. The formation of plasmid DNA/IL nanostructures was confirmed by measuring the zeta potential of plasmid DNA as well as plasmid DNA/IL nanostructures. The zeta potential of the nanostructures was positive, although plasmid DNA is negatively charged. The positive zeta potential is due to the complexation between plasmid DNA and positively charged ionic liquid [Bmim][PF6]. The ability of ionic liquid [Bmim][PF6] to protect plasmid DNA against ultrasonic shear stress was also investigated using an agarose gel electrophoretic assay and showed that ionic liquid stabilizes plasmid DNA against ultrasonication. The plasmid DNA and plasmid DNA/IL nanostructures were subjected to ultrasonic shear stress for different time periods and the biological functionality of pristine plasmid DNA (i.e., expression of the eGFP gene) as well as the self-assembled nanostructures was investigated in vitro using three different cell lines, COS7, HEK293 and HeLa. Ionic liquid [Bmim][PF6] protected the plasmid DNA against ultrasonic shear stress and also enhanced gene transfection efficiency in vitro. Furthermore, the cytotoxicity of ionic liquid [Bmim][PF6] was assayed in vitro using all three cell lines and the toxicity was very low. Therefore, the ionic liquid [Bmim][PF6] stabilizes plasmid DNA against ultrasonic shear stress and also enhances its in vitro delivery efficiency.

Iron Oxide Nanoparticles for Breast Cancer Theranostics.

BACKGROUND: Breast cancer is the second leading cause of death in women worldwide. The extremely fast rate of metastasis and ability to develop resistance mechanism to all the conventional drugs make them very difficult to treat which are the causes of high morbidity and mortality of breast cancer patients. Scientists throughout the world have been focusing on the early detection of breast tumor so that treatment can be started at the very early stage. Moreover, conventional treatment processes such as chemotherapy, radiotherapy, and local surgery suffer from various limitations including toxicity, genetic mutation of normal cells, and spreading of cancer cells to healthy tissues. Therefore, new treatment regimens with minimum toxicity to normal cells need to be urgently developed. METHODS: Iron oxide nanoparticles have been widely used for targeting hyperthermia and imaging of breast cancer cells. They can be conjugated with drugs, proteins, enzymes, antibodies or nucleotides to deliver them to target organs, tissues or tumors using external magnetic field. RESULTS: Iron oxide nanoparticles have been successfully used as theranostic agents for breast cancer both in vitro and in vivo. Furthermore, their functionalization with drugs or functional biomolecules enhance their drug delivery efficiency and reduces the systemic toxicity of drugs. CONCLUSION: This review mainly focuses on the versatile applications of superparamagnetic iron oxide nanoparticles on the diagnosis, treatment, and detecting progress of breast cancer treatment. Their wide application is because of their excellent superparamagnetic, biocompatible and biodegradable properties.

tRNA diversification among uncultured archeon clones.

Whole genome sequences (DNA sequences) of four uncultured archeon clones (1B6:CR626858.1, 4B7:CR626856.1, 22i07:JQ768096.1 and 19c08:JQ768095.1) were collected from NCBI BioSample database for the construction of digital data on tRNA. tRNAscan-SE 2.0 and ENDMEMO tools were used to identify and sketch tRNA structure as well as calculate Guanine-Cytosine (GC) percentage respectively. Eight true/functional tRNAs were identified from above 4 sequences which showed cove score greater than 20% with no variable loop. The tRNAs from the uncultured archeon clones were classified as Ala, Arg, Ile, Thr, Pro and Val type tRNA with cove score ranging from 34.22%-79.03%. The range of GC content was found 42.89%-56.91%; while tRNA contributed GC content ranging from 52%-64.86% to the total GC content in these sequences. The data fabricated in this study could be very useful for studying the diversity of tRNA among prokaryotes.

Intracellular delivery of universal proteins using a lysine headgroup containing cationic liposomes: deciphering the uptake mechanism.

An amino acid-based cationic lipid having a TFA counterion (trifluoroacetic acid counterion) in the lysine headgroup was used to deliver functional proteins into human cervical cancer cells, HeLa, in the presence of serum. Proteins used in the study were fluorescein isothiocyanate (FITC) labeled bovine serum albumin, mouse anti-F actin antibody [NH3], and goat anti mouse IgG conjugated with FITC. The formation of liposome/protein complexes was confirmed using native polyacrylamide gel electrophoresis. Furthermore, the complexes were characterized in terms of their size and zeta potential at different pH values and found to be responsive to changes in pH. The highest delivery efficiency of the liposome/albumin complexes was 99% at 37 °C. The liposomes effectively delivered albumin and antibodies as confirmed by confocal laser scanning microscopy (CLSM). Inhibition studies showed that the cellular uptake mechanism of the complexes was via caveolae-mediated endocytosis, and the proteins were subsequently released from either the early endosomes or the caveosomes as suggested by CLSM. Thus, lysine-based cationic liposomes can be a useful tool for intracellular protein delivery.

Cationic amino acid based lipids as effective nonviral gene delivery vectors for primary cultured neurons.

The delivery of specific genes into neurons offers a potent approach for treatment of diseases as well as for the study of neuronal cell biology. Here we investigated the capabilities of cationic amino acid based lipid assemblies to act as nonviral gene delivery vectors in primary cultured neurons. An arginine-based lipid, Arg-C3-Glu2C14, and a lysine-based lipid, Lys-C3-Glu2C14, with two different types of counterion, chloride ion (Cl-) and trifluoroacetic acid (TFA-), were shown to successfully mediate transfection of primary cultured neurons with plasmid DNA encoding green fluorescent protein. Among four types of lipids, we optimized their conditions such as the lipid-to-DNA ratio and the amount of pDNA and conducted a cytotoxicity assay at the same time. Overall, Arg-C3-Glu2C14 with TFA- induced a rate of transfection in primary cultured neurons higher than that of Lys-C3-Glu2C14 using an optimal weight ratio of lipid-to-plasmid DNA of 1. Moreover, it was suggested that Arg-C3-Glu2C14 with TFA- showed the optimized value higher than that of Lipofectamine2000 in experimental conditions. Thus, Arg-C3-Glu2C14 with TFA- is a promising candidate as a reliable transfection reagent for primary cultured neurons with a relatively low cytotoxicity.

Arginine-based cationic liposomes for efficient in vitro plasmid DNA delivery with low cytotoxicity.

BACKGROUND: Currently available gene delivery vehicles have many limitations such as low gene delivery efficiency and high cytotoxicity. To overcome these drawbacks, we designed and synthesized two cationic lipids comprised of n-tetradecyl alcohol as the hydrophobic moiety, 3-hydrocarbon chain as the spacer, and different counterions (eg, hydrogen chloride [HCl] salt or trifluoroacetic acid [TFA] salt) in the arginine head group. METHODS: Cationic lipids were hydrated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer to prepare cationic liposomes and characterized in terms of their size, zeta potential, phase transition temperature, and morphology. Lipoplexes were then prepared and characterized in terms of their size and zeta potential in the absence or presence of serum. The morphology of the lipoplexes was determined using transmission electron microscopy and atomic force microscopy. The gene delivery efficiency was evaluated in neuronal cells and HeLa cells and compared with that of lysine-based cationic assemblies and Lipofectamine™ 2000. The cytotoxicity level of the cationic lipids was investigated and compared with that of Lipofectamine™ 2000. RESULTS: We synthesized arginine-based cationic lipids having different counterions (ie, HCl-salt or TFA-salt) that formed cationic liposomes of around 100 nm in size. In the absence of serum, lipoplexes prepared from the arginine-based cationic liposomes and plasmid (p) DNA formed large aggregates and attained a positive zeta potential. However, in the presence of serum, the lipoplexes were smaller in size and negative in zeta potential. The morphology of the lipoplexes was vesicular. Arginine-based cationic liposomes with HCl-salt showed the highest transfection efficiency in PC-12 cells. However, arginine-based cationic liposomes with TFA salt showed the highest transfection efficiency in HeLa cells, regardless of the presence of serum, with very low associated cytotoxicity. CONCLUSION: The gene delivery efficiency of amino acid-based cationic assemblies is influenced by the amino acids (ie, arginine or lysine) present as the hydrophilic head group and their associated counterions.

Evaluation of the influence of ionization states and spacers in the thermotropic phase behaviour of amino acid-based cationic lipids and the transfection efficiency of their assemblies.

The influence of both the ionization states and the hydrocarbon chain spacer of a series of amino acid-based cationic lipids was evaluated in terms of gene delivery efficiency and cytotoxicity to the COS-7 cell line and compared with that of Lipofectamine 2000. We synthesized a series of amino acid-based cationic lipids with different ionization states (i.e., -NH(2), -NH(3)(+)Cl(-) or -NH(3)(+)TFA(-)) in the lysine head group and different hydrocarbon chain spacers (i.e., 0, 3, 5 or 7 carbon atoms) between the hydrophilic head group and hydrophobic moieties. In the 3-carbon series, the cationic assemblies formed a micellar structure in the presence of -NH(3)(+)Cl(-) and a vesicular structure both in the presence of -NH(2) and -NH(3)(+)TFA(-). Differential scanning calorimetry (DSC) data revealed a significantly lower (8.1°C) gel-to-liquid crystalline phase transition temperature for cationic assemblies bearing -NH(3)(+)TFA(-) when compared to their -NH(2) counterparts. Furthermore, the zeta potential of cationic assemblies having -NH(3)(+)TFA(-) in the hydrophilic head group was maximum followed by -NH(3)(+)Cl(-) and -NH(2) irrespective of their hydrocarbon chain spacer length. The gene delivery efficiency in relation to the ionization states of the hydrophilic head group was as follows: -NH(3)(+)TFA(-)>-NH(3)(+)Cl(-)>-NH(2).