Introduction

KCTD proteins represent an emerging class of proteins involved in fundamental physio-pathological pathways and pathological states, including neurological and neurodevelopmental processes, cancer, and genetic diseases^1-3. In the past decade, we have conducted extensive biochemical/biophysical characterizations of these proteins^4-6. However, the lack of structural data prevented a full understanding of their activities at the atomic level.

Methods

Here, by combining experimental (X-ray crystallography) and computational (molecular modeling/dynamics and structure prediction), we have extensively characterized the structural properties of all members of the KCTD family.

Results

Taking advantage of the recent advent of effective predictive approaches based on machine learning techniques (AlphaFold), we recently performed a detailed structural characterization of all members of the KCTD family. First, we demonstrated that the vast majority of these proteins share a structurally similar C-terminal domain despite the absence of sequence similarities. We generated a novel and comprehensive structure-based pseudo-phylogenetic tree that unraveled previously undetected similarities among the family⁷. A comprehensive analysis of the structural states of functional oligomers of all members of the family led us to identify reliable three-dimensional models⁸. Finally, we applied this approach to explore, at the atomic level, the Cul3 recognition of all KCTDs⁹.

Conclusions

Recently developed methodologies for protein structure prediction have made it possible to perform analyses on entire protein families. By combining experimental structural characterizations with effective predictions, we were able to gain insights into the structure of some KCTDs that will hold important implications for their biological function.

¹Teng et al. CNS Neurosci.Ther. 2019, 25; 887–902.

²Angrisani et al. Cell.Commun. Signal. 2021; 19, 56.

³Raymundo et al. J.Clin.Investig. 2023; e174138.

⁴Balasco et al. Biochim.Biophys.Acta 2014;1844(7):1289-98.

⁵Smaldone et al. PLoS One. 2015;10(5):e0126808.

⁶Balasco et al. Biomolecules. 2019;9(8):323.

⁷Esposito et al. Biomolecules. 2021;11(12):1862.

⁸Esposito et al. Int.J.Mol.Sci. 2022;23(21):13346.

⁹Balasco et al. Int.J.Mol.Sci. 2024;25(3):1881.

In the medical field, bone substitutes are essential for the reconstruction of bone fractures. Injectable and self-setting bone cements like magnesium phosphate (MPC) are critical for minimally invasive orthopedic surgeries. MPC offers beneficial bioresorption, rapid setting time, and high mechanical strength, rivaling that of classic calcium phosphates. However, its paste lacks appropriate cohesion and proper injectability.

This research explores the development of biocomposite cement based on MPC with a poly(vinyl alcohol) (PVA) hydrogel additive. The aim was to enhance the cement's usability by improving injectability and providing finer control of its setting process. The ceramic cement was synthesized via the reaction of magnesium oxide with potassium dihydrogen phosphate in an aqueous medium. To explore the effects of PVA, it was introduced in varying concentrations as the liquid phase of the cement. Additionally, different levels of a crosslinking agent were utilized to establish experimental groups. The mixture of these substances formed a biocomposite paste with self-setting properties. Our analysis involved measuring setting time and setting temperature, microstructure inspection, phase and chemical composition, static strength evaluation, a qualitative assessment of injectability, and a cytocompatibility test conducted on human osteoblast cells.

Via this study, we developed a technology for biocomposite cement combining MPC with PVA hydrogel. This novel material was characterized by high biocompatibility, reduced setting temperature, enhanced compressive strength, and appropriate injectability. Finally, it exhibits considerable potential for medical applications, especially within the fields of surgical orthopedics and traumatology.

Acknowledgments: This research was supported by the Gdańsk University of Technology by the DEC-3/2022/IDUB /III.4.3/Pu grant under the PLUTONIUM 'Excellence Initiative – Research University program.

Bone cements, as self-setting and injectable biomaterials, play a pivotal role in orthopedic and traumatological applications, serving crucial functions like bone loss restoration, implant fixation, and fracture stabilization. Recently, magnesium phosphate cements (MPC) have emerged as a noteworthy alternative to traditional calcium phosphate cements, acclaimed for their high mechanical strength, quick setting time, and optimal biodegradability. This research investigates the potential of gellan gum (GG) hydrogel as an enhancer for MPC, with a particular emphasis on improving their injectability.

The cement formulation under investigation was synthesized by combining a powder phase of magnesium oxide, potassium dihydrogen phosphate, and a cross-linking agent with a liquid phase, including a GG solution and a plasticizer, forming an injectable self-hardening paste. The properties of the resulting biocomposite cement, such as setting time and temperature, microstructure, mechanical strength, biodegradation rate, phase and chemical composition, injectability, and human osteoblast compatibility were evaluated.

The developed MPC+GG cement demonstrated an effective setting reaction at lower temperatures, reduced fragility, as well as improved injectability potential, enhancing its suitability for minimally invasive surgical procedures. Further, its high biocompatibility, appropriate porosity and adequate biodegradation rate were confirmed. Ultimately, the findings indicate the substantial applicability of this novel biocomposite cement in biomedical engineering.

Acknowledgments: This research was supported by the Gdańsk University of Technology by the DEC-3/2022/IDUB /III.4.3/Pu grant under the PLUTONIUM 'Excellence Initiative – Research University program.

The current study introduces a model in which the DNA sequence influences the structure of surrounding water in the nucleoplasm, proposing a mechanism where DNA's molecular structure imprints some of itself on surrounding water clusters during a continuous dynamic chromatin reorganization process within the cell nucleus. It is known that water is an integral part of DNA structure(s). In vivo, the DNA double helix is covered with a layer of hydration, impermeable to a variety of cations, which is located around these double helices. It consists of about 18-19 water molecules per nucleotide in B-DNA and different amounts for other DNA structures. These water molecules are specifically arranged into phosphate groups and bases. Our hypothesis suggests that the spatial arrangement of DNA shapes the organization of water. Therefore, the focus is, namely, on the role of water in the structural stability and special organization of nucleic DNA. Its role in multiple molecular recognition processes, which involve nucleic DNA, is addressed. We developed molecular models of the sheath of water surrounding the double helix; based on this, they developed predictions about the sequence-specificity of chromatin folding and tested these predictions using public functional genomics data and computational genomic approaches. The results provided support for the proposed models of sequence-specific chromatin folding. These findings suggest a structural mechanism of interaction between DNA and its aqueous environment, offering an additional sequence-specific structural basis for chromatin dynamics and function.

ABSTRACT

Introduction

Chandipura virus (CHPV) is a vesiculovirus that is a member of Rhabdviridae and is an encephalitic pathogen responsible for numerous epidemics in Central and Western India. The virus affects the brain and central nervous system mainly in children under 15 age of years, leading to neurological dysfunctions. Vectors that include sand flies, mosquitoes, and ticks are the main culprits in the transmission of CHPV. Five structural proteins (N, P, M, G, and L) encode the viral genome. The nucleocapsid protein N (N protein) encapsulates the viral genomic RNA in an RNase-resistant state, which plays a crucial role in the viral life cycle. Currently, no effective vaccine or therapeutics are available to treat the viral infection, and therefore efficient interventions are urgently needed.

Methods

The repurposing of drugs is one of the best possible ways to controlCHPV infections in India and other parts of the world. In this study, we used a structure-based virtual screening approach by using FDA-approved drugs against the nucleocapsid protein of CHPV. The docking process identified a few drug candidates, which showed potent binding affinity towards the N protein. We used the Schrödinger Desmond v3.0 module; to compute the relative binding energies of ligands, we used the premier mm-GBSA module.

Results

Based on a short molecular dynamics simulation and prime MM-GBSA analysis, we identified Adrabetadex, Hydroxypropyl betadex, Beta-1,2,3,4,6-penta-O-Galloyl-D-Glucopyranose, thio-maltohexaose, and Indium-III pentetreotide as potent drug candidates for CHPV.

Conclusion

Our computational results provide suggestions for in vitro and in vivo testing of these drugs against CHPV.

Introduction: Proteins that can bind several ligands at the same time are widespread in nature. We have developed an approach to study such interactions. The proposed method is based on the attachment of several binding domains to a single oligomeric backbone thus creating proteins with a specific number of binding sites.

Methods: To obtain proteins with different numbers of binding sites, we denatured and then renatured a mixture of the Sm-like protein from Sulfolobus acidocaldarius (SacSm) and the same protein but with the apical domain of the GroEL protein attached (ADGroEL_SacSm). By varying the ratio of the components we can change the number of apical domains on each resulting oligomer. After producing proteins with 1 to 7 binding domains, we determined the strength and stoichiometry of their interaction with the model non-native protein lactalbumin (LA). The interaction constants were assessed quantitatively by analyzing the results of SDS gel electrophoresis of the formed complexes.

Results: The data obtained suggest that the interaction between a single apical domain (AD) and non-native proteins is relatively weak and is not well-defined by our methods. However, when several ADs are closely located on a single protein backbone, non-native proteins may interact with several domains simultaneously, and the stability of this complex increases exponentially. LA cannot interact with more than two ADs at the same time, which is manifested by the lack of change in the dissociation constant as the number of binding sites increases. At the same time, its small size allows the binding of up to 4 molecules on the complete heptameric ADGroEL_SacSm ring.

Conclusion: We have developed a workable method for studying multivalent protein-protein interactions with a range of binding sites, from 1 to 7, which is essential for understanding the complexity of these interactions in biological systems.

This study explored the potential role of Clitoria ternatea (CT) flower in ameliorating endometrial pain (EP) through network pharmacology and experimental approaches. Phytochemicals of the CT flower were listed from the literature and databases, and 18 suitable actives were screened for bioavailability and drug likeness parameters using SwissADME. For these actives, 279 exclusive target genes were predicted using SwissTargetPrediction. Additionally, 939 exclusive genes for EP were acquired from the DisGenet and GeneCards databases. Ninety-one overlapping gene targets of CT and EP were listed, for which a Protein–Protein Interaction (PPI) network was constructed using STRING. The top three node proteins (SRC, ESR1, and PI3KR1) in the PPI network were identified through Cytoscape. Molecular docking analysis of the eighteen actives with the three target proteins showed strong binding interactions of Flavylium, kaempherol, and quercetin with all the targets, suggesting their involvement in EP relief. In addition, Gene Ontology (GO) functions analysis revealed 320 biological processes, 59 cellular components, and 107 molecular functions were enriched with the target genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses identified 106 KEGG pathways, including steroid hormone biosynthesis, endocrine resistance, and endometrial cancer pathways, which were significantly enriched with the target genes. The anti-inflammatory and analgesic effects of CT's methanolic extract (ME) were investigated through in vitro and in vivo assays. The ME exhibited 91.47% inhibition of heat-induced hemolysis compared to 92.87% by aspirin in the in vitro membrane stabilizing assay. The in vivo carrageenan-induced paw edema study revealed 65.28% inhibition of paw edema by ME compared to 80.38% inhibition by aceclofenac at the end of 4-hour treatment. The in vivo acetic acid-induced writhing test demonstrated analgesia by ME by 75.6% inhibition of writhing compared to 77.49% by aceclofenac. These findings suggest CT flower could be a potential natural remedy for EP, warranting further investigation in future studies.

Interest in biomaterials based on natural polymers has recently surged. The natural polymers are large molecular compounds, which can be obtained from living organisms or are produced by living organisms. Biopolymers typically exhibit properties highly desirable in tissue engineering, including biocompatibility, biodegradability, and minimal immune responses upon introduction into the human body. Chitosan, collagen, and silk fibroin are biopolymers commonly used in tissue engineering to obtain three-dimensional scaffolds [1]. They can be utilized as fillings for repairing small bone defects. However, materials obtained only from biopolymers or their mixtures may have insufficiently physicochemical properties. Therefore, there is a need to cross-link such materials [1]. The purpose of this work was to obtain and characterize 3D scaffolds based on chitosan, collagen, and silk fibroin modified with various cross-linking agents [2-4]. Collagen was obtained from young rat tail tendons, and silk fibroin was obtained from Bombyx mori cocoons. Materials were obtained using the lyophilization method, and their physicochemical properties, such as porosity, density, moisture content, and mechanical strength, were evaluated. Dialdehyde chitosan, dialdehyde starch, and glyoxal were used as modifiers of the three-component materials. Based on the results, it can be concluded that the properties of biopolymer materials depend on their weight ratio composition and the cross-linking agent. The study revealed that cross-linked materials exhibited a high swelling rate and sufficient porosity, rendering them suitable for tissue engineering applications. Mechanical properties vary depending on the composition of the blends. Novel biopolymeric composites based on collagen, chitosan, and silk fibroin, chemically cross-linked, hold potential for various biomedical applications, particularly in bone tissue regeneration.

[1] S. Grabska-Zielińska and A. Sionkowska, Materials 2021, 14, 1510.

[2] S. Grabska-Zielińska, et al., Int. J. Mol. Sci. 2021, 22, 3391.

[3] S. Grabska-Zielińska, et al., Materials 2020, 13, 3433.

[4] S. Grabska-Zielińska, et al., Polymers 2020, 12, 372.

Breast cancer (BC) is a complex disease driven by a combination of genetic mutations and epigenetic modifications. In particular, the overexpression of BET family proteins (BETs) has emerged as a key epigenetic aberration contributing to BC pathogenesis. CBL0137 (CBL), a small-molecule compound, has shown promise as an inhibitor of BETs in HeLa TI cells. In this study, we aimed to assess the anticancer effects of CBL in vitro and evaluate its impact on the expression of BETs in BC cells.

Cells of three subtypes of BC (MCF7, MDA-MB-231, SKBR3) were used in this study. Cytotoxic effects were analyzed using the MTT assay. Effects on cell cycle and apoptosis were assessed using FACS with PI and FITC-Annexin staining. The level of BETs (BRD2, BRD3, BRD4) was determined by Western blotting.

CBL demonstrated a significant reduction in BС cell viability with an IC50 value of approximately 1 μM for all cell lines after 72h of exposure and 20 μM after 24h. CBL treatment resulted in an increase in cells in the G2/M phase in MCF7 and SKBR3 cells after 24h and 72h of action, as well as in MDA-MB-231 cells after 24h. In MCF7 cells, the influence of CBL led to apoptotic changes characterized by a slight elevation in the early apoptotic population. Treatment of MDA-MB-231 cells with CBL resulted in a decrease in the expression of BRD2, BRD3, and BRD4 proteins, while treatment of MCF7 cells led to a reduction in BRD3 and BRD4 protein levels. No significant changes in the amount of BET proteins were observed in SKBR3 cells.

In conclusion, the presented data offer valuable insights into the mechanisms of action of CBL, providing a basis for further investigation into its therapeutic potential in BC treatment. This research was funded by the RSF (no. 21-75-10163).

Single-walled carbon nanotubes (SWCNTs) are referred to as a promising material for biocompatible devices and novel medicines. In these applications, contact between SWCNTs and blood should be expected. Serum albumin is the most abundant globular protein in mammalian blood, so it is of great importance to study the interactions of SWCNTs with major blood components such as serum albumin, as contact between them is possible with the incorporation of SWCNTs into common practice. The radiotracer technique with [³H] is highly sensitive and enables the quantification of biomolecules in complexes with nanomaterials without inducing significant changes in the biomolecule structure.

We simulated the interaction of bovine serum albumin (BSA) as a model protein with SWCNTs moderately functionalized with fluorine with Gromacs software. Partial atom charges and bond lengths were calculated with the semi-empirical quantum chemistry method PM7 with MOPAC2016 software. Thermostat, barostat and basic simulations were carried out with the Berendsen algorithm. The simulation time was 100 ns. The dynamics of changes in the secondary structure of BSA were simulated. The quantity of BSA bonded with SWCNTs was estimated with the use of the radiotracer technique, with [³H]BSA obtained with the tritium thermal activation technique.

We found that the dominant interactions between BSA and SWCNTs are hydrophobic. The fluorine atoms in the SWCNTs become involved in hydrogen bonds with His 534, Thr 539 and Ala 583 in the BSA. The mean total energy of the Coulomb and Van der Waals interactions is -364 ± 9 kJ/mol, as determined by gmx energy. The maximum absorption of BSA on the SWCNTs is 740 mg/mg, which is consistent in all the simulations and leads to the ζ-potential of the BSA–SWCNT complex changing from -10 to -16 mV as the adsorption increases.

The research was carried out using equipment from the shared research facilities of HPC computing resources at Lomonosov Moscow State University.