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.

The purpose of this study was to evaluate the in vivo biological potential of new azetidine-2-one derivatives of ferulic acid (6a−f). First, the in vivo acute toxicity of azetidine-2-one derivatives of ferulic acid on Swiss white mice was investigated and, based on the obtained results, it can be stated that the studied derivatives belong to compounds with moderate toxicity. The in vivo anti-inflammatory potential of these derivatives was determined in a model of acute inflammation induced by carrageenan in rats and in a chronic inflammation model induced in rats using the granuloma test. In the acute inflammation model, all the studied compounds had a maximum anti-inflammatory effect 24 h after administration, which suggests that these compounds may be classified, from a pharmacokinetic point of view, in the category of long-acting compounds. The most active compound in the series was found to be compound 6b. In the case of the chronic inflammation model, it was observed that the stud... [more]

A laser ablation process assisted by the feedback of a sensor with chaotic electronic modulation is reported. A synchronous bistable logic circuit was analyzed for switching optical signals in a laser-processing technique. The output of a T-type flip-flop configuration was employed in the photodamage of NiO films. Multiphotonic effects involved in the ablation threshold were evaluated by a vectorial two-wave mixing method. A photoinduced thermal phenomenon was identified as the main physical mechanism responsible for the nonlinearity of index under nanosecond irradiation at 532 nm wavelength. Comparative experiments for destroying highly transparent human cells were carried out. Potential applications for developing hierarchical functions yielding laser-induced controlled explosions with immediate applications for biomedical photothermal processes can be contemplated.

Tissue engineering (TE) is one of the most challenging fields of research since it provides current alternative protocols and materials for the regeneration of damaged tissue. The success of TE has been mainly related to the right selection of nano-sized biocompatible materials for the development of matrixes, which can display excellent anatomical structure, functionality, mechanical properties, and histocompatibility. Today, the research community has paid particular attention to zein as a potential biomaterial for TE applications and nanotechnological approaches. Considering the properties of zein and the advances in the field, there is a need to reviewing the current state of the art of using this natural origin material for TE and nanotechnological applications. Therefore, the goal of this review paper is to elucidate the latest (over the last five years) applications and development works in the field, including TE, encapsulations of drugs, food, pesticides and bandaging for exte... [more]

Carbon dioxide is the most influential gas in greenhouse gasses and its amount in the atmosphere reached 412 µmol/mol in August 2020, which increased rapidly, by 48%, from preindustrial levels. A brand-new chemical industry, namely organic chemistry and catalysis science, must be developed with carbon dioxide (CO2) as the source of carbon. Nowadays, many techniques are available for controlling and removing carbon dioxide in different chemical processes. Since the utilization of CO2 as feedstock for a chemical commodity is of relevance today, this study will focus on how to increase CO2 solubility in culture media used for growing microbes. In this work, the CO2 solubility in a different medium was investigated. Sodium hydroxide (NaOH) and monoethanolamine (MEA) were added to the culture media (3.0 g/L dipotassium phosphate (K2HPO4), 0.2 g/L magnesium chloride (MgCl2), 0.2 g/L calcium chloride (CaCl2), and 1.0 g/L sodium chloride (NaCl)) for growing microbes in order to observe the dif... [more]

Lab-on-a-Disc (LoaD) biosensors are increasingly a promising solution for many biosensing applications. In the search for a perfect match between point-of-care (PoC) microfluidic devices and biosensors, the LoaD platform has the potential to be reliable, sensitive, low-cost, and easy-to-use. The present global pandemic draws attention to the importance of rapid sample-to-answer PoC devices for minimising manual intervention and sample manipulation, thus increasing the safety of the health professional while minimising the chances of sample contamination. A biosensor is defined by its ability to measure an analyte by converting a biological binding event to tangible analytical data. With evolving manufacturing processes for both LoaDs and biosensors, it is becoming more feasible to embed biosensors within the platform and/or to pair the microfluidic cartridges with low-cost detection systems. This review considers the basics of the centrifugal microfluidics and describes recent developm... [more]

(1) Background: shape, dimension, hemodynamics, and hemocompatibility are just a few of the several challenging key points that must be addressed in designing any suitable solution for the ventricular chamber of mechanical circulatory support devices. A preliminary evaluation of different geometries of bellow-like ventricular chambers is herein proposed. The chambers were made with a polycarbonate urethane that is acknowledged to be a hemocompatible polymer. (2) Methods: an explicit dynamic computational analysis was performed. The actuation of the three chambers was simulated without the presence of an internal fluid. Maximum stress and strain values were identified, as well as the most critical regions. Geometric changes were checked during simulated motion to verify that the dimensional constraints were satisfied. (3) Results: one chamber appeared to be the best solution compared to the others, since its dimensional variations were negligible, and effective stresses and strains did... [more]

The study aims to prepare a smart copolymeric for controlled delivery of Galantamine hydrobromide. The synthesis of the hydrogel was executed through free radical polymerization using HPMC (Hydroxypropyl methylcellulose) and pectin as polymers and acrylic acid as monomer. Cross-linking was performed by methylene bisacrylamide (MBA). HPMC-pectin-co-acrylic acid hydrogel was loaded with Galantamine hydrobromide (antidementia drug) as a model drug for treatment of Alzheimer based dementia. Formulated hydrogels (SN1−SN9) were characterized for Fourier transform-infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction, and energy dispersive X-ray. Drug loading efficiency, gel fraction, measurements of porosity, and tensile strength were reported. Swelling and release studies were performed at pH 1.2 and 7.4. Drug liberation mechanism was evaluated by applying different release kinetic models. Galantamine hydrobromide was released from prepared... [more]

The activated sludge respiration inhibition test and the luminescent bacteria test with Vibrio fischeri are important bacterial test systems for evaluation of the toxicity of chemical compounds. These test systems were further optimized to result in better handling, reliability and sensitivity. Concerning the Vibrio fischeri test, media components such as yeast extract and bivalent cation concentrations like Ca2+ and Mg2+ were optimized. The cultivation, storage conditions and reactivation process of the stored bacteria were also improved, which enabled simpler handling and led to good reproducibility. Additionally, the respiration inhibition test with a prolonged incubation time was further analyzed using different chlorinated phenols as reference compounds. It could be stated that a longer incubation period significantly improved the sensitivity of the test system.

Improving the hygienic status of feed ingredients by biotechnological processes as fermentation is of the greatest concern. This preliminary study aimed to investigate whether there are relevant effects of fermented liquid feed (FLF) on the survival of potential pathogens in vitro. The feed (fresh basis) consisted of 50% rye, 30% rapeseed extracted meal, 10% barley and 10% wheat. Glass bottles were filled about 14.1 g water (38 °C) containing the diluted starter culture and feed (8.81 g). Fermentation led to high levels of lactate (5−7% of dry matter), low pH values (<4.0) and low levels of acetic acid (<1% of dry matter) in the FLF. The survival rate of pathogens added, such as Salmonella enterica serovar Typhimurium, Escherichia coli and Clostridium perfringens after 6 h of controlled fermentation, was significantly reduced (<2 log10 CFU/g). The counts of Candida krusei in FLF at 3 h and 6 h post inoculation remained almost unchanged regardless of the incubation time. Even a... [more]

Bone injuries that arise from trauma, cancer treatment, or infection are a major and growing global challenge. An increasingly ageing population plays a key role in this, since a growing number of fractures are due to diseases such as osteoporosis, which place a burden on healthcare systems. Current reparative strategies do not sufficiently consider cell-substrate interactions that are found in healthy tissues; therefore, the need for more complex models is clear. The creation of in vitro defined 3D microenvironments is an emerging topographically-orientated approach that provides opportunities to apply knowledge of cell migration and differentiation mechanisms to the creation of new cell substrates. Moreover, introducing biofunctional agents within in vitro models for bone regeneration has allowed, to a certain degree, the control of cell fate towards osteogenic pathways. In this research, we applied three methods for functionalizing spatially-confined electrospun artificial microenvi... [more]

In recent years, polymeric hydrogels (PolyHy) have been extensively explored for their applications in biomedicine as biosensors, in tissue engineering, diagnostic processes, and drug release. The physical and chemical properties of PolyHy indicate their potential use in regulating drug delivery. Calcineurin inhibitors, particularly cyclosporine (CsA) and tacrolimus (TAC), are two important immunosuppressor drugs prescribed upon solid organ transplants. Although these drugs have been used since the 1970s to significantly increase the survival of transplanted organs, there are concerns regarding their undesirable side effects, primarily due to their highly variable concentrations. In fact, calcineurin inhibitors lead to acute and chronic toxicities that primarily cause adverse effects such as hypertension and nephrotoxicity. It is suggested from the evidence that the encapsulation of calcineurin inhibitors into PolyHy based on polysaccharides, specifically alginate (Alg), offers effecti... [more]

High cell density cultivation (HCDC) is developed for the production of microbial biomasses and their products. They must be produced from high concentrations of substrate, e.g., glucose or sucrose. In batch culture, a high concentration of those sugars >40−50% (w/v) cannot efficiently be utilized because of a dissolved O2 limitation causing the Crabtree effect that produces toxic by-products, i.e., ethanol and/or acetate, that inhibit cell growth. To prevent this effect, the HCDC is conducted with the fed-batch strategies. However, it has many disadvantages, i.e., complicated operations. To overcome those problems, this study was designed to use a new, efficient C-source (carbon source) substrate, namely dextrin, an oligomer of glucose. It can be utilized by yeast at a very high concentration of ~100 g/L although using just batch cultivation. As it is gradually hydrolyzed to release glucose molecules and gradually assimilated into the cells as “fed-batch at the cell level” (FBC), it p... [more]

Background: Despite the widespread use of newer basal insulins, Natural Protamine Hagedorn (NPH) insulin still represents a well-established basal formulation with its long history of use, featuring the native form of human insulin. However, NPH insulin exhibits an undesirable peak within hours after a single subcutaneous (s.c.) injection, which may lead to hypoglycemia followed by insufficient basal insulin delivery. This may be attributed to the s.c. enzyme activities degrading the protamine in NPH microcrystals. Methods: A thermogelling block copolymer Pluronic® F127 (PF127) was utilized as a protective carrier for NPH microcrystals and as a modulator for insulin release from NPH. NPH insulin-loaded PF127 gel was prepared with varying concentrations of the polymer (15−25%) under mild conditions. The formulations were characterized for their gelling temperature, morphology, gel erosion, and in vitro insulin release, with trypsin concentrations up to 5 U/mL. Results: Scanning electron... [more]

Both molecular analyses and culture-dependent isolation were combined to investigate the diversity of sulfate-reducing prokaryotes and explore their role in sulfides production in full-scale anaerobic digesters (Marrakech, Morocco). At global scale, using 16S rRNA gene sequencing, Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Synergistetes, and Euryarchaeota were the most dominant phyla. The abundance of Archaea (3.1−5.7%) was linked with temperature. The mcrA gene ranged from 2.18 × 105 to 1.47 × 107 gene copies.g−1 of sludge. The sulfate-reducing prokaryotes, representing 5% of total sequences, involved in sulfides production were Peptococcaceae, Syntrophaceae, Desulfobulbaceae, Desulfovibrionaceae, Syntrophobacteraceae, Desulfurellaceae, and Desulfobacteraceae. Furthermore, dsrB gene ranged from 2.18 × 105 to 1.92 × 107 gene copies.g−1 of sludge. The results revealed that exploration of diversity and function of sulfate-reducing bacteria may play a key role in decreasin... [more]

The sodium triple-quantum (TQ) magnetic resonance (MR) signal created by interactions of sodium ions with macromolecules has been demonstrated to be a valuable biomarker for cell viability. The aim of this study was to monitor a cellular response using the sodium TQ signal during inhibition of Na/K-ATPase in living cancer cells (HepG2). The cells were dynamically investigated after exposure to 1 mM ouabain or K+-free medium for 60 min using an MR-compatible bioreactor system. An improved TQ time proportional phase incrementation (TQTPPI) pulse sequence with almost four times TQ signal-to-noise ratio (SNR) gain allowed for conducting experiments with 12−14 × 106 cells using a 9.4 T MR scanner. During cell intervention experiments, the sodium TQ signal increased to 138.9 ± 4.1% and 183.4 ± 8.9% for 1 mM ouabain (n = 3) and K+-free medium (n = 3), respectively. During reperfusion with normal medium, the sodium TQ signal further increased to 169.2 ± 5.3% for the ouabain experiment, while i... [more]