The study investigated in situ biosynthesis of albumin capped 5-fluorouracil (5-FU) loaded gold nanoparticles (NPs) using bacterial extract for enhanced efficacy against MCF-7 and in silico prediction using a GastroPlus® software. The optimized formulations were characterized for morphology, size, zeta potential, drug loading (%DL) and entrapment (), compatibility, in vitro drug release, in vitro hemolysis, cellular toxicity and apoptosis studies. The results exhibited highly dispersed albumin capped mono-metallic stable NPs. Spherical size, negative zeta potential and polydispersity index were in range of 38.25−249.62 nm, 18.18−29.87 mV and 0.11−0.283, respectively. F11, F7 and F3 showed a progressive increase in %DL and with increased concentration of the cellular lysate (100% > 50% > 10%). The drug release was relatively extended over 48 h as compared to drug solution (96.64% release within 5 h). The hemolysis result ensured hemocompatibility (<14%) at the explored concentration.... [more]

Adipocyte-specific transcription factors and antioxidants are considered the best target of obesity. Aruncus dioicus var. kamtschaticus (A. dioicus, Samnamul) is easily available owing to edible and inexpensive. However, the anti-adipogenic effects of the underlying mechanism of A. dioicus extract (ADE) have not yet been reported. In the present study, we evaluate anti-adipogenic pathway in 3T3-L1 adipocytes, antioxidant activities and quantified phenolics using high-performance liquid chromatography of ADE. The results revealed ADE had reduced adipocyte differentiation (0.72-fold vs. MDI (media of differentiation) control), triglyceride (TG; 0.50-fold vs. MDI control, p < 0.001), and total cholesterol contents (0.77-fold vs. MDI control) by regulating adipocyte-specific transcription factors (C/EBPα, PPARγ, and SREBP1) and their downstream mRNA (AdipoQ, Ap2, SREBP1-c, and FAS) levels. Furthermore, ADE has higher total phenol and flavonoid contents and scavenging assay in the DPPH a... [more]

We use a system biology approach to translate the interaction of Bacillus Calmette-Gurin (BCG) + interleukin 2 (IL-2) for the treatment of bladder cancer into a mathematical model. The main goal of this research is to predict the outcome of BCG + IL-2 treatment combinations. We examined whether the delay effect caused by the proliferation of tumor antigen-specific effector cells after the immune system destroys BCG-infected urothelium cells after BCG and IL-2 immunotherapy influences success in bladder cancer treatment. To do this, we introduce a system of differential equations where the variables are the main participants in the immune response after BCG installations to fight cancer: the number of tumor cells, BCG cells, immune cells, and cytokines involved in the tumor-immune response. The relevant parameters describing the dynamics of the system are taken from a variety of biological, clinical literature and estimated using the mathematical models. We examine the local stability a... [more]

Exosomes are defined as a type of extracellular vesicle released when multivesicular bodies of the endocytic pathway fuse with the plasma membrane. They are characterized by their role in extracellular communication, partly due to their composition, and present the ability to recognize and interact with cells from the immune system, enabling an immune response. Their targeting capability and nanosized dimensions make them great candidates for cancer therapy. As chemotherapy is associated with cytotoxicity and multiple drug resistance, the use of exosomes targeting capabilities, able to deliver anticancer drugs specifically to cancer cells, is a great approach to overcome these disadvantages. The objective is to assess treatment efficiency in reducing tumor cells, as well as overall safety and response by cancer carriers. So far, results show exosomes as a promising therapeutic strategy in the fight against cancer. This review summarizes the characteristics and composition of exosomes,... [more]

Many venomous species, especially snakes, contain a variety of secreted phospholipases A2 that contribute to venom toxicity and prey digestion. We characterized a novel highly toxic phospholipase A2 of group II, WaPLA2-II, from the snake venom of Saudi Walterinnesia aegyptia (W. aegyptia). The enzyme was purified using a reverse phase C18 column. It is a monomeric protein with a molecular weight of approximately 14 kDa and an NH2-terminal amino acid sequence exhibiting similarity to the PLA2 group II enzymes. WaPLA2-II, which contains 2.5% (w/w) glycosylation, reached a maximal specific activity of 1250 U/mg at pH 9.5 and 55 °C in the presence of Ca2+ and bile salts. WaPLA2-II was also highly stable over a large pH and temperature range. A strong correlation between antimicrobial and indirect hemolytic activities of WaPLA2 was observed. Additionally, WaPLA2-II was found to be significantly cytotoxic only on cancerous cells. However, chemical modification with para-Bromophenacyl bromide... [more]

The modern view of the mechanism of intercellular movement of viruses is based largely on data from the study of the tobacco mosaic virus (TMV) 30-kDa movement protein (MP). The discovered properties and abilities of TMV MP, namely, (a) in vitro binding of single-stranded RNA in a non-sequence-specific manner, (b) participation in the intracellular trafficking of genomic RNA to the plasmodesmata (Pd), and (c) localization in Pd and enhancement of Pd permeability, have been used as a reference in the search and analysis of candidate proteins from other plant viruses. Nevertheless, although almost four decades have passed since the introduction of the term “movement protein” into scientific circulation, the mechanism underlying its function remains unclear. It is unclear why, despite the absence of homology, different MPs are able to functionally replace each other in trans-complementation tests. Here, we consider the complexity and contradictions of the approaches for assessment of the... [more]

Over the past decades, anaerobic biotechnology is commonly used for treating high-strength wastewaters from different industries. This biotechnology depends on interactions and co-operation between microorganisms in the anaerobic environment where many pollutants’ transformation to energy-rich biogas occurs. Properties of wastewater vary across industries and significantly affect microbiome composition in the anaerobic reactor. Methanogenic archaea play a crucial role during anaerobic wastewater treatment. The most abundant acetoclastic methanogens in the anaerobic reactors for industrial wastewater treatment are Methanosarcina sp. and Methanotrix sp. Hydrogenotrophic representatives of methanogens presented in the anaerobic reactors are characterized by a wide species diversity. Methanoculleus sp., Methanobacterium sp. and Methanospirillum sp. prevailed in this group. This work summarizes the relation of industrial wastewater composition and methanogen microbial communities present in... [more]

The spike protein of the pandemic human corona virus is essential for its entry into human cells. In fact, most neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) are directed against the Virus-surface exposed spike protein, making it the antigen of choice for use in vaccines and diagnostic tests. In the current pandemic context, global demand for spike proteins has rapidly increased and could exceed hundreds of grams to kilograms annually. Coronavirus spikes are large heavily glycosylated homo-trimeric complexes, with inherent instability. The poor manufacturability now threatens the availability of these proteins for vaccines and diagnostic tests. Here, we outline scalable, Good Manufacturing Practice (GMP) compliant, and chemically defined processes for the production of two cell-secreted stabilized forms of the trimeric spike proteins (Wuhan and D614G variant). The processes are chemically defined and based on clonal suspension-CHO cell po... [more]

Liposomes are well-investigated drug or gene delivery vehicles for chemotherapy, used by taking advantage of their biocompatibility and biodegradability. A central question on the construction of intracellular liposomal delivery systems is to entrap the liposomes of interest in the highly acidic and proteolytic endosomal environment. In the other words, it is essential that the liposomes release a therapeutic drug into the cytosol before they are degraded in the endosome. As a strategy to enhance the endosome escape, the self-lytic liposomes with acidic pH-selective membrane active polypeptide are considered highly effective. Here, an acidic pH-selective membrane-lytic polypeptide (LPE) and its retro isomer (rLPE) were designed, and then their membrane-lytic activities against EggPC liposomes were determined. It was noticed that the rLPE polypeptide showed an increase in activity compared with the LPE polypeptide. Furthermore, the rLPE polypeptide was conjugated to liposomes via a flex... [more]

Tuna blood (TB) was subjected to enzymatic hydrolysis. The effects of the relationship of hydrolysis time (30−180 min) and enzyme concentration (0.5−3.0% w/w protein) on the degree of hydrolysis (DH), yield, antioxidant and angiotensin-I-converting enzyme (ACE) inhibitory activities were determined. The response surface methodology (RSM) showed that TB hydrolysis’s optimum conditions were hydrolysis for 180 min and Alcalase, Neutrase or Flavourzyme at 2.81%, 2.89% or 2.87% w/w protein, respectively. The hydrolysates with good DH (40−46%), yield (3.5−4.6%), the IC50 of DPPH (0.8−1.6 mg/mL) and ABTS (1.0−1.4 mg/mL) radical scavenging activity, ferric reducing antioxidant power (FRAP) value (0.28−0.65 mmol FeSO4/g) and IC50 of ACE inhibitory activity (0.15−0.28 mg/mL) were obtained with those conditions. The TB hydrolysate using Neutrase (TBHN) was selected for characterization in terms of amino acid composition, peptide fractions and sensory properties. The essential, hydrophobic and hyd... [more]

Dried blood spot (DBS) technology has become a promising utility for the transportation and storage of biological fluids aimed for the subsequent clinical analysis. The basis of the DBS method is the adsorption of the components of a biological sample onto the surface of a membrane carrier, followed by drying. After drying, the molecular components of the biosample (nucleic acids, proteins, and metabolites) can be analyzed using modern omics, immunological, or genomic methods. In this work, we investigated the safety of proteins on a membrane carrier by tryptic components over time and at different temperatures (+4, 0, 25 °C) and storage (0, 7, 14, and 35 days). It was shown that the choice of a protocol for preliminary sample preparation for subsequent analytical molecular measurements affects the quality of the experimental results. The protocol for preliminary preparation of a biosample directly in a membrane carrier is preferable compared to the protocol with an additional stage of... [more]

In 2016, infectious microbes were one of the leading causes of death, especially in developing countries. Puriton® is a mineral mixture consisting of biotite, kaolinite, montmorillonite, serpentine, clinochlore, and vermiculite, and evaluated antimicrobial activity in vitro and safety in vivo. Nine pathogens and opportunistic bacteria, namely Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Alcaligenes faecalis, Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus, Mycobacterium smegmatis, and Bacillus subtilis, and the two viruses Zika and Influenza A/Duck/MN/1525/81 were used. A 26-week oral repeated safety study of Puriton® was conducted. Puriton® suppressed the bacterial proliferation, with a minimum proliferative rate of 91.1% in B. subtilis ATCC6633. The virucidal efficacy of Puriton® against Zika virus after 4 h and 18 h of contact time was significant in all groups treated with Puriton®. Twenty-six-week repeated oral administration of Puriton® was c... [more]

The aim of this study was to synthesize, characterize, and evaluate neoteric imine compounds for antimicrobial activity and hemocompatibility. Four compounds were synthesized using 3-thiophene carboxaldehyde, ethanol, amine, and acetic acid. The compounds were characterized using nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD). A solubility study was conducted with various solvents and surfactants at 40 °C. An in vitro antimicrobial assay was performed against bacterial and fungal strains to determine the zone of inhibition and minimum inhibitory concentrations. Finally, an in vitro hemolysis study was conducted using rat erythrocytes. The structure of the compounds was confirmed by NMR, FTIR corroborated their functional group attributes, DSC determined their enthalpies of fusion and fusion temperatures, and PXRD confirmed their crystalline nature. These compound... [more]

 In the present report, we provide the results of the molecular biology studies of spiroheterocyclic hybrids, where the derivatives are found to possess potential anticancer activity towards cancer cells. A series of spiroxindole−pyrrolidine heterocyclic hybrids were evaluated for cell viability and proliferation against HepG2 cancer cells at concentrations in the range of 12.5−200 µg/mL over two different time periods of 24 and 48 h. In addition, the highly active compounds were also verified for their behavior towards noncancer cells (L929 cells), and it was found that the tested derivatives were not aggressive due to the observation of only limited cell loss, as compared to the cancer cells. Further analysis of the observed toxicity mechanism showed the apoptotic pathway was mediated by oxidative stress, with the involvement of caspases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in severe damage to the respiratory system. With no specific treatment to date, it is crucial to identify potent inhibitors of SARS-CoV-2 Chymotrypsin-like protease (3CLpro) that could also modulate the enzymes involved in the respiratory damage that accompanies SARS-CoV-2 infection. Here, flavones isolated from Scutellaria baicalensis (baicalein, baicalin, wogonin, norwogonin, and oroxylin A) were studied as possible compounds in the treatment of SARS-CoV-2 and SARS-CoV-2-induced acute lung injuries. We used structural bioinformatics and cheminformatics to (i) identify the critical molecular features of flavones for their binding activity at human and SARS-CoV-2 enzymes; (ii) predict their drug-likeness and lead-likeness features; (iii) calculate their pharmacokinetic profile, with an emphasis on toxicology; (iv) predict their pharmacodynamic profiles, with the identification of their human body targets... [more]

The insect cell-baculovirus expression vector system is an established method for large scale recombinant adeno-associated virus (rAAV) production, largely due to its scalability and high volumetric productivities. During rAAV production it is critical to monitor process parameters such as Spodoptera frugiperda (Sf9) cell concentration, infection timing, and cell harvest viabilities since they can have a significant influence on rAAV productivity and product quality. Herein we developed the use of dielectric spectroscopy as a process analytical technology (PAT) tool used to continuously monitor the production of rAAV in 2 L stirred tank bioreactors, achieving enhanced control over the production process. This study resulted in improved manufacturing robustness through continuous monitoring of cell culture parameters, eliminating sampling needs, increasing the accuracy of infection timing, and reliably estimating the time of harvest. To increase the accuracy of baculovirus infection tim... [more]

Artificial construction of spherical protein assemblies has attracted considerable attention due to its potential use in nanocontainers, nanocarriers, and nanoreactors. In this work, we demonstrate a novel strategy to construct peptide nanocapsules (artificial viral capsids) decorated with enzymes via interactions between His-tag and Ni-NTA. A β-annulus peptide derived from the tomato bushy stunt virus was modified with Ni-NTA at the C-terminus, which is directed toward the exterior surface of the artificial viral capsid. The β-annulus peptide bearing Ni-NTA at the C-terminus self-assembled into capsids of about 50 nm in diameter. The Ni-NTA-displayed capsids were complexed with recombinant horseradish peroxidase (HRP) with a C-terminal His-tag which was expressed in Escherichia coli. The β-annulus peptide-HRP complex formed spherical assemblies whose sizes were 30−90 nm, with the ζ-potential revealing that the HRP was decorated on the outer surface of the capsid.

Improved productivity of the two most commonly used cell lines in the biopharmaceutical industry, such as human embryonic kidney 293 (HEK293) and Chinese hamster ovary (CHO), could reduce production costs and increase manufacturing capacity. One method for increasing protein productivity is the addition of antioxidants during the cell culture process. In this study, we examined the effect of sialic acid (SA) on one HEK293 cell line and two CHO cell lines. The addition of SA to HEK293 cell led to a higher viable cell density (VCD), viability (Via), and a lower lactate content in the later stage of cultures. Further results showed that SA reduced the reactive oxygen species (ROS), improved cell viability, reduced lactate production, and increased antibody expression by more than 20% in the later stage of the two CHO cell lines cultures. Besides, an optimized dose of SA had no significant effect on acidic variants level aggregation level, N-linked glycosylation pattern, and SA content on... [more]

Controlling ice nucleation during lyophilization of parenteral drug products increases the homogeneity of critical quality attributes, such as residual moisture, across drug product batches and shortens lyophilization cycle time. In the present study, we compare three mechanistically different techniques to control ice nucleation during the freezing step of lyophilization, which are referred to as “depressurization”, “partial vacuum”, and “ice fog” techniques. The techniques are compared with respect to their operational limitations and challenges. Installation considerations are also discussed. Using the aforementioned nucleation techniques, we investigated a monoclonal antibody formulation and an enzyme formulation at different protein concentrations using feasible nucleation temperatures and different vial formats and fill volumes. Samples were compared for solid state properties and other critical quality attributes on stability. When nucleated at the same temperature, the three te... [more]

The potentiality of a microalgal-bacterial culture system was explored in bioremediating wastewater while generating biomass for biodiesel production. A pre-determined optimal activated sludge and microalgal ratio was adopted and cultivation performance was evaluated in both synthetic and municipal wastewater media for nitrogen removal along with biomass and lipid generation for biodiesel production. The microalgal-bacterial consortium grown in the municipal wastewater medium produced higher biomass and lipid yields than those in the synthetic wastewater medium. The presence of trace elements in the municipal wastewater medium, e.g., iron and copper, contributed to the upsurge of biomass, thereby leading to higher lipid productivity. Both the microbial cultures in the synthetic and municipal wastewater media demonstrated similar total nitrogen removal efficiencies above 97%. However, the nitrification and assimilation rates were relatively higher for the microbial culture in the munici... [more]

A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. In this study, we implemented a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification, and nano-scale qPCR based on a commercially available microfluidic chip. Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of this microfluidic RT-qPCR by 1000-fold, enabling detection below 1 copy/µL. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible and quantitative detection of SARS-CoV-2 ov... [more]

Hydrogen peroxide (H2O2) is an important oxidizing molecule that regulates the metabolisms of aerobic organisms. Redox signaling comprises physiological oxidative stress (eustress), while excessive oxidative stress causes damage to molecules. The main enzymatic generators of H2O2 are nicotinamide adenine dinucleotide phosphate oxidases or NADPH oxidases (NOXs) and mitochondrial respiratory chains, as well as various oxidases. The NOX family is constituted of seven enzyme isoforms that produce a superoxide anion (O2−), which can be converted to H2O2 by superoxide dismutase or spontaneously. H2O2 passes through the membranes by some aquaporins (AQPs), known as peroxyporins. It diffuses through cells and tissues to initiate cellular effects, such as proliferation, the recruitment of immune cells, and cell shape changes. Therefore, it has been proposed that H2O2 has the same importance as Ca2+ or adenosine triphosphate (ATP) to act as modulators in signaling and the metabolism. The present... [more]

There is an increasing interest in designing fibrillogenesis modulators for treating amyloid β (Aβ)-peptide-associated diseases. The use of Aβ fragment peptides and their derivatives, as well as nonpeptidyl natural products, is one promising approach to prevent Aβ fibrillation. In this study, we demonstrate that tandem-homodimers (TDs) of a β-sheet-forming short peptide in which the amino acid sequence is duplicated in series and joined via an amino alkanoic acid linker of different chain lengths, preventing the random-to-β structural transition of the original monomer. Ape5-TD, containing 5-amino pentanoate, most potently prevented this transition for at least five days by generating disordered aggregates with reduced tryptic stability. The linkers in the TDs generated this inhibitory activity, probably due to their bent conformations and hydrophobicity, appropriate for accommodating and twisting the monomers, resulting in irregular arrangements of the peptides. The present study coul... [more]

Acellular assays of oxidative potential (OP) induced by ambient particulate matters (PMs) are of great significance in screening for toxicity in PMs. In this review, several typical OP measurement techniques, including the respiratory tract lining fluid assay (RTLF), ascorbate depletion assay (AA), dithiothreitol assay (DTT), chemiluminescent reductive acridinium triggering (CRAT), dichlorofluorescin assay (DCFH) and electron paramagnetic/spin resonance assay (EPR/ESR) are discussed and their sensitivity to different PMs species composition, PMs size distribution and seasonality is compared. By comparison, the DTT assay tends to be the preferred method providing a more comprehensive measurement with transition metals and quinones accumulated in the fine PMs fraction. Specific transition metals (i.e., Mn, Cu, Fe) and quinones are found to contribute OPDTT directly whereas the redox properties of PMs species may be changed by the interactions between themselves. The selection of the appr... [more]

Buerger’s disease or Thromboangiitis Obliterans (TAO) is a nonatherosclerotic segmental vascular disease which affects small and medium arteries and veins in the upper and lower extremities. Based on pathological findings, TAO can be considered as a distinct form of vasculitis that is most prevalent in young male smokers. There is no definitive cure for this disease as therapeutic modalities are limited in number and efficacy. Surgical bypass has limited utility and 24% of patients will ultimately require amputation. Recently, studies have shown that therapeutic angiogenesis and immunomodulatory approaches through the delivery of stem cells to target tissues are potential options for ischemic lesion treatment. In this review, we summarize the current knowledge of TAO treatment and provide an overview of stem cell-based treatment modalities.