Chitosan is a polymer that is extensively used to prepare nanoparticles (NPs) with tailored properties for applications in many fields of human activities. Among them, targeted drug delivery, especially when cancer therapy is the main interest, is a major application of chitosan-based NPs. Due to its positive charges, chitosan is used to produce the core of the NPs or to cover NPs made from other types of polymers, both strategies aiming to protect the carried drug until NPs reach the target sites and to facilitate the uptake and drug delivery into these cells. A major challenge in the design of these chitosan-based NPs is the formation of a protein corona (PC) upon contact with biological fluids. The composition of the PC can, to some extent, be modulated depending on the size, shape, electrical charge and hydrophobic/hydrophilic characteristics of the NPs. According to the composition of the biological fluids that have to be crossed during the journey of the drug-loaded NPs towards the target cells, the surface of these particles can be changed by covering their core with various types of polymers or with functionalized polymers carrying some special molecules, that will preferentially adsorb some proteins in their PC. The PC’s composition may change by continuous processes of adsorption and desorption, depending on the affinity of these proteins for the chemical structure of the surface of NPs. Beside these, in designing the targeted drug delivery NPs one can take into account their toxicity, initiation of an immune response, participation (enhancement or inhibition) in certain metabolic pathways or chemical processes like reactive oxygen species, type of endocytosis of target cells, and many others. There are cases in which these processes seem to require antagonistic properties of nanoparticles. Products that show good behavior in cell cultures may lead to poor in vivo results, when the composition of the formed PC is totally different. This paper reviews the physico-chemical properties, cellular uptake and drug delivery applications of chitosan-based nanoparticles, specifying the factors that contribute to the success of the targeted drug delivery. Furthermore, we highlight the role of the protein corona formed around the NP in its intercellular fate.

Chitin is an abundant biopolymer found mainly in the exoskeleton of crustaceans and insects. The degradation of chitin using chitinases is one way to address the accumulation of chitin waste streams in the environment, and research has therefore focused on the identification, improvement and expression of suitable enzymes. Here we describe the production, purification and characterization of Bacillus licheniformis chitinase A in the Pichia pastoris expression system. Optimal enzyme activity occurred at pH 4.0-5.0 and within the temperature range 50-60 °C. With colloidal chitin as the substrate, the Km (2.307 mM) and Vmax (0.024 mM min-1) of the enzyme were determined using a 3,5-dinitrosalicylic acid assay. The degradation products of colloidal chitin and hexa-N-acetylchitohexaose were compared by thin-layer chromatography. The activity of the glycosylated enzyme produced in P. pastoris was compared with the in vitro deglycosylated and aglycosylated version produced in Escherichia coli. We showed that the glycosylated chitinase was more active than the deglycosylated and aglycosylated variants.

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

The aim of this research was to investigate the bioremediation conditions of copper in synthetic water. In the present study, copper ions accumulation efficiency was determined using various genetically modified strains of Saccharomyces cerevisiae (EBY100, INVSc1, BJ5465, and GRF18), Pichia pastoris (X-33, KM71H), Escherichia coli (XL10 Gold, DH5α, and six types of BL21 (DE3)), and Escherichia coli BL21 (DE3) OverExpress expressing two different peroxidases. Viability tests of yeast and bacterial strains showed that bacteria are viable at copper concentrations up to 2.5 mM and yeasts up to 10 mM. Optical emission spectrometry with inductively coupled plasma analysis showed that the tolerance of bacterial strains on media containing 1 mM copper was lower than the tolerance of yeast strains at the same copper concentration. The E. coli BL21 RIL strain had the best copper accumulation efficiency (4.79 mg/L of culture normalized at an optical density of 1.00), which was 1250 times more efficient than the control strain. The yeast strain S. cerevisiae BJ5465 was the most efficient in copper accumulation out of a total of six yeast strains used, accumulating over 400 times more than the negative control strain. In addition, E. coli cells that internally expressed recombinant peroxidase from Thermobifida fusca were able to accumulate 400-fold more copper than cells that produced periplasmic recombinant peroxidases.

DNA analysis plays a crucial role in forensic investigations, helping in criminal cases, missing persons inquiries, and archaeological research. This study focuses on the DNA concentration in different skeletal elements to improve human identification efforts. Ten cases of unidentified skeletal remains brought to the Institute of Forensic Medicine in Timisoara, Romania, underwent DNA analysis between 2019 and 2023. The results showed that teeth are the best source for DNA extraction as they contain the highest concentration of genetic material, at 3.68 ng/µL, compared to the petrous temporal bone (0.936 ng/µL) and femur bone (0.633 ng/µL). These findings highlight the significance of teeth in forensic contexts due to their abundant genetic material. Combining anthropological examination with DNA analysis enhances the understanding and precision of identifying human skeletal remains, thus advancing forensic science. Selecting specific skeletal elements, such as the cochlea or teeth, emerges as crucial for reliable genetic analyses, emphasizing the importance of careful consideration in forensic identification procedures. Our study concludes that automated DNA extraction protocols without liquid nitrogen represent a significant advancement in DNA extraction technology, providing a faster, more efficient, and less labor-intensive method for extracting high-quality DNA from damaged bone and tooth samples.

Bor, Krivelj, and Bela Rivers in Eastern Serbia belong to the watershed of Timok River, which is a tributary of Danube River. These Rivers flow near to the largest mining complex in Republic of Serbia. Rivers Bosneag, Radimna and Nera from the Romanian side in Caras Severin County near to Moldova Noua are also tributary of Danube and flow in area which is affected by spreading of dust from the abandoned flotation tailing of copper mine. This study includes environmental monitoring of the surface waters in considered Romania-Serbia cross border area during 2019-2020. Chemical analyses confirmed pollution mostly with heavy metals (Fe, Mn, Cu, Zn, As, Ni, Pb, Cd) closer to mines while with larger distance pollution decrease. The measured values were compared with Serbian and Romanian legislation which confirmed moderate and high pollution depending on category of surface waters. Measured Cd concentrations were of 9.51 - 5375 μg L-1 for the IV category of River water close to Bor mine. The main anthropogenic source in Eastern Serbia was Bor mine and smelter.