Biosynthesis of Functional Silver Nanoparticles Using Callus and Hairy Root Cultures of Aristolochia manshuriensis.

This study delves into the novel utilization of Aristolochia manshuriensis cultured cells for extracellular silver nanoparticles (AgNPs) synthesis without the need for additional substances. The presence of elemental silver has been verified using energy-dispersive X-ray spectroscopy, while distinct surface plasmon resonance peaks were revealed by UV-Vis spectra. Transmission and scanning electron microscopy indicated that the AgNPs, ranging in size from 10 to 40 nm, exhibited a spherical morphology. Fourier-transform infrared analysis validated the abilty of A. manshuriensis extract components to serve as both reducing and capping agents for metal ions. In the context of cytotoxicity on embryonic fibroblast (NIH 3T3) and mouse neuroblastoma (N2A) cells, AgNPs demonstrated varying effects. Specifically, nanoparticles derived from callus cultures exhibited an IC50 of 2.8 µg/mL, effectively inhibiting N2A growth, whereas AgNPs sourced from hairy roots only achieved this only at concentrations of 50 µg/mL and above. Notably, all studied AgNPs' treatment-induced cytotoxicity in fibroblast cells, yielding IC50 values ranging from 7.2 to 36.3 µg/mL. Furthermore, the findings unveiled the efficacy of the synthesized AgNPs against pathogenic microorganisms impacting both plants and animals, including Agrobacterium rhizogenes, A. tumefaciens, Bacillus subtilis, and Escherichia coli. These findings underscore the effectiveness of biotechnological methodologies in offering advanced and enhanced green nanotechnology alternatives for generating nanoparticles with applications in combating cancer and infectious disorders.

Preparation and Characterization of Hydrogel Films and Nanoparticles Based on Low-Esterified Pectin for Anticancer Applications.

Prospective adjuvant anticancer therapy development includes the establishing of drug delivery systems based on biocompatible and biodegradable carriers. We have designed films and nanoparticles (NPs) based on low-esterified pectin hydrogel using the ionic gelation method. We investigated morphology, nanomechanical properties, biocompatibility and anticancer activity. Hydrogel films are characterized by tunable viscoelastic properties and surface nanoarchitectonics through pectin concentration and esterification degree (DE), expressed in variable pore frequency and diameter. An in vitro study showed a significant reduction in metabolic activity and the proliferation of the U87MG human glioblastoma cell line, probably affected via the adhesion mechanism. Glioma cells formed neurosphere-like conglomerates with a small number of neurites when cultured on fully de-esterified pectin films and they did not produce neurites on the films prepared on 50% esterified pectin. Pectin NPs were examined in terms of size distribution and nanomechanical properties. The NPs' shapes were proved spherical with a mean diameter varying in the range of 90-115 nm, and a negative zeta potential from -8.30 to -7.86 mV, which indicated their stability. The NPs did not demonstrate toxic effect on cells or metabolism inhibition, indicating good biocompatibility. Nanostructured biomaterials prepared on low-esterified pectins could be of interest for biomedical applications in adjuvant anticancer therapy and for designing drug delivery systems.

Enhanced Production of Nitrogenated Metabolites with Anticancer Potential in Aristolochia manshuriensis Hairy Root Cultures.

Aristolochia manshuriensis is a relic liana, which is widely used in traditional Chinese herbal medicine and is endemic to the Manchurian floristic region. Since this plant is rare and slow-growing, alternative sources of its valuable compounds could be explored. Herein, we established hairy root cultures of A. manshuriensis transformed with Agrobacterium rhizogenes root oncogenic loci (rol)B and rolC genes. The accumulation of nitrogenous secondary metabolites significantly improved in transgenic cell cultures. Specifically, the production of magnoflorine reached up to 5.72 mg/g of dry weight, which is 5.8 times higher than the control calli and 1.7 times higher than in wild-growing liana. Simultaneously, the amounts of aristolochic acids I and II, responsible for the toxicity of Aristolochia species, decreased by more than 10 fold. Consequently, the hairy root extracts demonstrated pronounced cytotoxicity against human glioblastoma cells (U-87 MG), cervical cancer cells (HeLa CCL-2), and colon carcinoma (RKO) cells. However, they did not exhibit significant activity against triple-negative breast cancer cells (MDA-MB-231). Our findings suggest that hairy root cultures of A. manshuriensis could be considered for the rational production of valuable A. manshuriensis compounds by the modification of secondary metabolism.

How to Develop Drug Delivery System Based on Carbohydrate Nanoparticles Targeted to Brain Tumors.

Brain tumors are the most difficult to treat, not only because of the variety of their forms and the small number of effective chemotherapeutic agents capable of suppressing tumor cells, but also limited by poor drug transport across the blood-brain barrier (BBB). Nanoparticles are promising drug delivery solutions promoted by the expansion of nanotechnology, emerging in the creation and practical use of materials in the range from 1 to 500 nm. Carbohydrate-based nanoparticles is a unique platform for active molecular transport and targeted drug delivery, providing biocompatibility, biodegradability, and a reduction in toxic side effects. However, the design and fabrication of biopolymer colloidal nanomaterials have been and remain highly challenging to date. Our review is devoted to the description of carbohydrate nanoparticle synthesis and modification, with a brief overview of the biological and promising clinical outcomes. We also expect this manuscript to highlight the great potential of carbohydrate nanocarriers for drug delivery and targeted treatment of gliomas of various grades and glioblastomas, as the most aggressive of brain tumors.

Research Topics of the Bioinformatics of Gene Regulation.

The study of gene expression regulation raises the challenge of developing bioinformatics tools and algorithms, demanding data integration [...].

Kidney-Related Function of Mitochondrial Protein Mitoregulin.

A small protein, Mitoregulin (Mtln), localizes in mitochondria and contributes to oxidative phosphorylation and fatty acid metabolism. Mtln knockout mice develop obesity on a high-fat diet, demonstrating elevated cardiolipin damage and suboptimal creatine kinase oligomerization in muscle tissue. Kidneys heavily depend on the oxidative phosphorylation in mitochondria. Here we report kidney-related phenotypes in aged Mtln knockout mice. Similar to Mtln knockout mice muscle mitochondria, those of the kidney demonstrate a decreased respiratory complex I activity and excessive cardiolipin damage. Aged male mice carrying Mtln knockout demonstrated an increased frequency of renal proximal tubules' degeneration. At the same time, a decreased glomerular filtration rate has been more frequently detected in aged female mice devoid of Mtln. An amount of Mtln partner protein, Cyb5r3, is drastically decreased in the kidneys of Mtln knockout mice.

Mitoregulin Contributes to Creatine Shuttling and Cardiolipin Protection in Mice Muscle.

Small peptides compose a large share of the mitochondrial proteome. Mitoregulin (Mtln) is a mitochondrial peptide known to contribute to the respiratory complex I functioning and other processes in mitochondria. In our previous studies, we demonstrated that Mtln knockout mice develop obesity and accumulate triglycerides and other oxidation substrates in serum, concomitant with an exhaustion of tricarboxylic acids cycle intermediates. Here we examined the functional role of Mtln in skeletal muscles, one of the major energy consuming tissues. We observed reduced muscle strength for Mtln knockout mice. Decrease of the mitochondrial cardiolipin and concomitant increase in monolysocardiolipin concentration upon Mtln inactivation is likely to be a consequence of imbalance between oxidative damage and remodeling of cardiolipin. It is accompanied by the mitochondrial creatine kinase octamer dissociation and suboptimal respiratory chain performance in Mtln knockout mice.

Nanomechanical Signatures in Glioma Cells Depend on CD44 Distribution in IDH1 Wild-Type but Not in IDH1R132H Mutant Early-Passage Cultures.

Atomic force microscopy (AFM) recently burst into biomedicine, providing morphological and functional characteristics of cancer cells and their microenvironment responsible for tumor invasion and progression, although the novelty of this assay needs to coordinate the malignant profiles of patients' specimens to diagnostically valuable criteria. Applying high-resolution semi-contact AFM mapping on an extended number of cells, we analyzed the nanomechanical properties of glioma early-passage cell cultures with a different IDH1 R132H mutation status. Each cell culture was additionally clustered on CD44+/- cells to find possible nanomechanical signatures that differentiate cell phenotypes varying in proliferative activity and the characteristic surface marker. IDH1 R132H mutant cells compared to IDH1 wild-type ones (IDH1wt) characterized by two-fold increased stiffness and 1.5-fold elasticity modulus. CD44+/IDH1wt cells were two-fold more rigid and much stiffer than CD44-/IDH1wt ones. In contrast to IDH1 wild-type cells, CD44+/IDH1 R132H and CD44-/IDH1 R132H did not exhibit nanomechanical signatures providing statistically valuable differentiation of these subpopulations. The median stiffness depends on glioma cell types and decreases according to the following manner: IDH1 R132H mt (4.7 mN/m), CD44+/IDH1wt (3.7 mN/m), CD44-/IDH1wt (2.5 mN/m). This indicates that the quantitative nanomechanical mapping would be a promising assay for the quick cell population analysis suitable for detailed diagnostics and personalized treatment of glioma forms.

Nanomechanical and Morphological AFM Mapping of Normal Tissues and Tumors on Live Brain Slices Using Specially Designed Embedding Matrix and Laser-Shaped Cantilevers.

Cell and tissue nanomechanics has been intriguingly introduced into biomedical research, not only complementing traditional immunophenotyping and molecular analysis, but also bringing unexpected new insights for clinical diagnostics and bioengineering. However, despite the progress in the study of individual cells in culture by atomic force microscopy (AFM), its application for mapping live tissues has a number of technical limitations. Here, we elaborate a new technique to study live slices of normal brain tissue and tumors by combining morphological and nanomechanical AFM mapping in high throughput scanning mode, in contrast to the typically utilized force spectroscopy mode based on single-point probe application. This became possible due to the combined use of an appropriate embedding matrix for vibratomy and originally modified AFM probes. The embedding matrix composition was carefully developed by regulating the amounts of agar and collagen I to reach optimal viscoelastic properties for obtaining high-quality live slices that meet AFM requirements. AFM tips were rounded by irradiating them with focused nanosecond laser pulses, while the resulting tip morphology was verified by scanning electron microscopy. Live slices preparation and AFM investigation take only 55 min and could be combined with a vital cell tracer analysis or immunostaining, thus making it promising for biomedical research and clinical diagnostics.

Cell and Tissue Nanomechanics: From Early Development to Carcinogenesis.

Cell and tissue nanomechanics, being inspired by progress in high-resolution physical mapping, has recently burst into biomedical research, discovering not only new characteristics of normal and diseased tissues, but also unveiling previously unknown mechanisms of pathological processes. Some parallels can be drawn between early development and carcinogenesis. Early embryogenesis, up to the blastocyst stage, requires a soft microenvironment and internal mechanical signals induced by the contractility of the cortical actomyosin cytoskeleton, stimulating quick cell divisions. During further development from the blastocyst implantation to placenta formation, decidua stiffness is increased ten-fold when compared to non-pregnant endometrium. Organogenesis is mediated by mechanosignaling inspired by intercellular junction formation with the involvement of mechanotransduction from the extracellular matrix (ECM). Carcinogenesis dramatically changes the mechanical properties of cells and their microenvironment, generally reproducing the structural properties and molecular organization of embryonic tissues, but with a higher stiffness of the ECM and higher cellular softness and fluidity. These changes are associated with the complete rearrangement of the entire tissue skeleton involving the ECM, cytoskeleton, and the nuclear scaffold, all integrated with each other in a joint network. The important changes occur in the cancer stem-cell niche responsible for tumor promotion and metastatic growth. We expect that the promising concept based on the natural selection of cancer cells fixing the most invasive phenotypes and genotypes by reciprocal regulation through ECM-mediated nanomechanical feedback loop can be exploited to create new therapeutic strategies for cancer treatment.

Novel assay to measure chromosome instability identifies Punica granatum extract that elevates CIN and has a potential for tumor- suppressing therapies.

Human artificial chromosomes (HACs) have provided a useful tool to study kinetochore structure and function, gene delivery, and gene expression. The HAC propagates and segregates properly in the cells. Recently, we have developed an experimental high-throughput imaging (HTI) HAC-based assay that allows the identification of genes whose depletion leads to chromosome instability (CIN). The HAC carries a GFP transgene that facilitates quantitative measurement of CIN. The loss of HAC/GFP may be measured by flow cytometry or fluorescence scanning microscope. Therefore, CIN rate can be measured by counting the proportion of fluorescent cells. Here, the HAC/GFP-based assay has been adapted to screen anticancer compounds for possible induction or elevation of CIN. We analyzed 24 cytotoxic plant extracts. Punica granatum leaf extract (PLE) indeed sharply increases CIN rate in HT1080 fibrosarcoma cells. PLE treatment leads to cell cycle arrest, reduction of mitotic index, and the increased numbers of micronuclei (MNi) and nucleoplasmic bridges (NPBs). PLE-mediated increased CIN correlates with the induction of double-stranded breaks (DSBs). We infer that the PLE extract contains a component(s) that elevate CIN, making it a candidate for further study as a potential cancer treatment. The data also provide a proof of principle for the utility of the HAC/GFP-based system in screening for natural products and other compounds that elevate CIN in cancer cells.

A Novel C1q Domain-Containing Protein Isolated from the Mollusk Modiolus kurilensis Recognizing Glycans Enriched with Acidic Galactans and Mannans.

C1q domain-containing (C1qDC) proteins are a group of biopolymers involved in immune response as pattern recognition receptors (PRRs) in a lectin-like manner. A new protein MkC1qDC from the hemolymph plasma of Modiolus kurilensis bivalve mollusk widespread in the Northwest Pacific was purified. The isolation procedure included ammonium sulfate precipitation followed by affinity chromatography on pectin-Sepharose. The full-length MkC1qDC sequence was assembled using de novo mass-spectrometry peptide sequencing complemented with N-terminal Edman's degradation, and included 176 amino acid residues with molecular mass of 19 kDa displaying high homology to bivalve C1qDC proteins. MkC1qDC demonstrated antibacterial properties against Gram-negative and Gram-positive strains. MkC1qDC binds to a number of saccharides in Ca2+-dependent manner which characterized by structural meta-similarity in acidic group enrichment of galactose and mannose derivatives incorporated in diversified molecular species of glycans. Alginate, κ-carrageenan, fucoidan, and pectin were found to be highly effective inhibitors of MkC1qDC activity. Yeast mannan, lipopolysaccharide (LPS), peptidoglycan (PGN) and mucin showed an inhibitory effect at concentrations three orders of magnitude greater than for the most effective saccharides. MkC1qDC localized to the mussel hemal system and interstitial compartment. Intriguingly, MkC1qDC was found to suppress proliferation of human adenocarcinoma HeLa cells in a dose-dependent manner, indicating to the biomedical potential of MkC1qDC protein.

Advances in the Understanding of Skin Cancer: Ultraviolet Radiation, Mutations, and Antisense Oligonucleotides as Anticancer Drugs.

Skin cancer has always been and remains the leader among all tumors in terms of occurrence. One of the main factors responsible for skin cancer, natural and artificial UV radiation, causes the mutations that transform healthy cells into cancer cells. These mutations inactivate apoptosis, an event required to avoid the malignant transformation of healthy cells. Among these deadliest of cancers, melanoma and its 'younger sister', Merkel cell carcinoma, are the most lethal. The heavy toll of skin cancers stems from their rapid progression and the fact that they metastasize easily. Added to this is the difficulty in determining reliable margins when excising tumors and the lack of effective chemotherapy. Possibly the biggest problem posed by skin cancer is reliably detecting the extent to which cancer cells have spread throughout the body. The initial tumor is visible and can be removed, whereas metastases are invisible to the naked eye and much harder to eliminate. In our opinion, antisense oligonucleotides, which can be used in the form of targeted ointments, provide real hope as a treatment that will eliminate cancer cells near the tumor focus both before and after surgery.

Immune state correlates with histopathological level and reveals molluscan health in populations of Modiolus kurilensis by integral health index (IHI).

Quantitative analysis of the histopathological and immune parameters of bivalve Modiolus kurilensis collected from water areas with different level of ecotoxicological stress was performed. Significant differences between samples from polluted and non-polluted sites were revealed for total haemocyte count; percentage of agranulocytes; size and internal complexity of agranulocytes and granulocytes; phagocytic activity; percentage of NBT-positive cells; hemolytic activity and plasma protein concentration; percentage of the optical density of haemolymph major polypeptide bands at 55 kDa, 78 kDa, and 124 kDa; concretion coverage area in the kidney tubules; thickness of the tubular basement membrane; nephrocyte shape; and karyopyknosis of the kidneys; and hypervacuolisation; necrosis; karyopyknosis; haemocyte infiltration; fibrosis; and invasion of the digestive gland. Analysis of the global histopathological condition index based on the weighted indices also revealed that both the digestive gland and kidneys showed significantly greater histopathological changes in the bivalves collected from polluted water. Bivalve histopathology is an established tool in aquatic toxicology. However, it reflects a morphological picture of change, which, as a rule, can be clearly recorded only at the later stages of pathology, and in some cases, indicates an adaptation to stressors within the physiological norm. In this respect, a promising and highly sensitive biomarker of the functional state of bivalves, in terms of norm and pathology as well as their habitat, is the evaluation of immune status in combination with morphological changes. However, the use of different methods and scales of assessment and the diagnosis of biomarkers, characterised by different profiles of the stress response, makes it difficult to compare the results of different studies. We propose a reliable and powerful system for assessing the physiological state of bivalve molluscs, expressed in the integral health index (IHI) and based on the standardisation of the numerical values for all parameters that have significant differences between animals collected from impacted and non-impacted water areas. In our study, IHI calculated in three variants (for histopathological parameters, for immunological parameters, and in combination) showed the most significant differences in each of the cases, but the strongest difference (-4.07) was in calculating the total IHI, which included both the immune and histopathological parameters (p = 0.00005).

The fate of larval flagellated cells during metamorphosis of the sponge Halisarca dujardini.

Sponge larval flagellated cells have been known to form the external layer of larva, but their subsequent fate and morphogenetic role are still unclear. It is actually impossible to follow flagellated cell developmental fate unless a specific marker is found. We used percoll density gradient fractionation to separate different larval cell types of Halisarca dujardini (Demospongiae, Halisarcida). A total of 5 fractions were obtained which together contained all cell types. Fraction 1 contained about 100% FC and its polypeptide composition was very different to that of the other fractions. Of all larval cell types, flagellated cells displayed the lowest in vitro aggregation capacity. We raised a polyclonal antibody against a 68 kDa protein expressed by larval flagellated cells. Its specificity was tested on total protein extract from adult sponges by Western blotting and proved to be suitable for immunofluorescence. By means of double immunofluorescence using both this polyclonal antibody and commercial anti-tubulin antibodies, we studied the distribution of the 68 kDa protein in larval flagellated cells and its fate at successive stages of metamorphosis. In juvenile sponges just after metamorphosis the choanocytes and the upper pinacoderm were labelled with both antibodies. In larval flagellated cells, the 68 kDa protein was found all over the cytoplasm appearing as granules, while in adult sponges, it was present in the apical part of choanocytes in the vicinity of collars. Direct participation of the larval flagellated cells in the development of definitive structures was demonstrated.