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Polymer nanocomposites obtain many new functional properties by adding appropriate nanoparticles, making them more suitable for applications in tissue engineering. The subjects of the presented research are nanocomposites composed of poly(e-caprolactone) (PCL) matrix and carbon nanofibers as nanoadditions. Each of the tested materials contained a different type of fibrous nanoparticles: first, carbon nanofibers in the initial state (CNFs), and the second, carbon nanofibers after oxidative functionalization (CNFs-f). Both materials, PCL/CNFs and PCL/CNFs-f, were confirmed to be characterized by high bioactivity, and at the same time induced osteoblast proliferation. The aim of this work was to resolve the details of the complex relations between osteoblast-like U-2 OS cells and material, that denote the nanoaddition specificity. The 2D Raman correlation spectroscopy technique revealed that in both of the composites the key component of their interactions with the cells was the carbonous domains of the materials. In the PCL/CNFs the G-mode of the CNFs correlate asynchronously with Amide I of the U-2 OS cells, whereas, the D1-band is mostly engaged into interaction with the side chain amino acids in the case of the PCL/CNFs-f nanocomposite.

The objects of this research were two types of polymer nanocomposites: poly(e-caprolactone)/multiwalled carbon nanotubes (PCL/MWCNTs) and poly(e-caprolactone)/functionalized multiwalled carbon nanotubes (PCL/MWCNTs-f) (without and with oxidative treatment of MWCNTs prior to nanocomposite production, respectively). These nanocomposites are promising materials for medical and tissue engineering applications. The cell proliferation test, performed by culturing of osteoblast-like U-2 OS cells line (human bone osteosarcoma epithelial cells) on the surface of tested nanocomposites, confirmed their high biocompatibility. In order to distinguish the nature of cells-material interactions the two-dimensional (2D) Raman correlation spectroscopy technique was used. It revealed more developed changes in the proteins of the cells cultured on the PCL/MWCNTs-f compare to PCL/MWCNTs, as the conformation β of amide I emerged in the asynchronous map of PCL/MWCNTs-f. Moreover, it was confirmed that the functionalization of MWCNTs leads to a different pattern of nanocomposite interaction with cells, for PCL/MWCNTs-f the formed cross-peaks indicate the creation of combined domains of polymer with cell proteins. Whereas for PCL/MWCNTs it was predominantly the nanotubes’ amorphous component D1 that induced the cross-peaks formation.

USP2a is a deubiquitinating protease that rescues its target proteins from destruction by the proteasome by reversing the process of protein ubiquitination. USP2a shows oncogenic properties in vivo and has been found to be a specific activator of cyclin D1. Many types of cancers are addicted to cyclin D1 expression. Targeting USP2a is a promising strategy for cancer therapy but little progress has been made in the field of inhibition of USP2a. Using NMR-based fragment screening and biophysical binding assays, we have discovered small molecules that bind to USP2a. Iterations of fragment combination and structure-driven design identified two 5-(2-thienyl)-3-isoxazoles as the inhibitors of the USP2a-ubiquitin protein-protein interaction. The affinity of these molecules for the catalytic domain of USP2a parallels their ability to interfere with USP2a binding to ubiquitin in vitro. Altogether, our results establish the 5-(2-thienyl)-3-isoxazole pharmacophore as an attractive starting point for lead optimization.

Hyperinsulinaemic hypoglycaemia (HH) is also classically referred to as "nesidioblastosis". Heterogeneous clinical manifestation of the disease causes risk of late diagnosis or even misdiagnosis. In infants and children, it can lead to serious and permanent damage to the central nervous system, which leads to the manifesting mental retardation. HH is characterised by unregulated insulin secretion from pancreatic β-cells. This effect has been correlated with nine genes: ABCC8, KCNJ11, GCK, GLUD-1, HADH1, SLC16A1, HNF4A, HNF1A, and UCP2. Mutations in these genes were found in approximately 48% of cases. The genetic background of the remaining cases is unknown. Understanding the genetic basis of familial hyperinsulinism has changed the early look at the disease. It has allowed for the differentiation of specific types of the disease. Depending on which of the nine disease-associated loci bears a pathogenic mutation, they differ in phenotype and pattern of inheritance. This review provides a brief overview of the genetic mechanisms of HH and its possible clinical presentations.

Blockade of the PD-1/PD-L1 immune checkpoint pathway with monoclonal antibodies has provided significant advances in cancer treatment. The antibody-based immunotherapies carry a number of disadvantages such as the high cost of the antibodies, their limited half-life, and immunogenicity. Development of small-molecule PD-1/PD-L1 inhibitors that could overcome these drawbacks is slow because of the incomplete structural information for this pathway. The first chemical PD-1/PD-L1 inhibitors have been recently disclosed by Bristol-Myers Squibb. Here we present NMR and X-ray characterization for the two classes of these inhibitors. The X-ray structures of the PD-L1/inhibitor complexes reveal one inhibitor molecule located at the center of the PD-L1 homodimer, filling a deep hydrophobic channel-like pocket between two PD-L1 molecules. Derivatives of (2-methyl-3-biphenylyl)methanol exhibit the structures capped on one side of the channel, whereas the compounds based on [3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methanol induce an enlarged interaction interface that results in the open "face-back" tunnel through the PD-L1 dimer.

Antibodies targeting the PD-1/PD-L1 immune checkpoint achieved spectacular success in anticancer therapy in the recent years. In contrast, no small molecules with cellular activity have been reported so far. Here we provide evidence that small molecules are capable of alleviating the PD-1/PD-L1 immune checkpoint-mediated exhaustion of Jurkat T-lymphocytes. The two optimized small-molecule inhibitors of the PD-1/PD-L1 interaction, BMS-1001 and BMS-1166, developed by Bristol-Myers Squibb, bind to human PD-L1 and block its interaction with PD-1, when tested on isolated proteins. The compounds present low toxicity towards tested cell lines and block the interaction of soluble PD-L1 with the cell surface-expressed PD-1. As a result, BMS-1001 and BMS-1166 alleviate the inhibitory effect of the soluble PD-L1 on the T-cell receptor-mediated activation of T-lymphocytes. Moreover, the compounds were effective in attenuating the inhibitory effect of the cell surface-associated PD-L1. We also determined the X-ray structures of the complexes of BMS-1001 and BMS-1166 with PD-L1, which revealed features that may be responsible for increased potency of the compounds compared to their predecessors. Further development may lead to the design of an anticancer therapy based on the orally delivered immune checkpoint inhibition.

The tumor suppressor protein p53, the "guardian of the genome", is inactivated in nearly all cancer types by mutations in the TP53 gene or by overexpression of its negative regulators, oncoproteins MDM2/MDMX. Recovery of p53 function by disrupting the p53-MDM2/MDMX interaction using small-molecule antagonists could provide an efficient nongenotoxic anticancer therapy. Here we present the syntheses, activities, and crystal structures of the p53-MDM2/MDMX inhibitors based on the 1,4,5-trisubstituted imidazole scaffold which are appended with aliphatic linkers that enable coupling to bioactive carriers. The compounds have favorable properties at both biochemical and cellular levels. The most effective compound 19 is a tight binder of MDM2 and activates p53 in cancer cells that express the wild-type p53, leading to cell cycle arrest and growth inhibition. Crystal structures reveal that compound 19 induces MDM2 dimerization via the aliphatic linker. This unique dimerization-binding mode opens new prospects for the optimization of the p53-MDM2/MDMX inhibitors and conjugation with bioactive carriers.

Streptococcus mutans (MS) and its biotype I are the strains most frequently found in dental plaque of young children. Our results indicate that in children pyruvate kinase (PK) activity increases significantly in dental plaque, and this corresponds with caries progression. The MS strains isolated in this study or their main glycolytic metabolism connected with PK enzymes might be useful risk factors for studying the pathogenesis and target points of novel therapies for dental caries. The relationship between PK activity, cariogenic biofilm formation and selected biotypes occurrence was studied. S. mutans dental plaque samples were collected from supragingival plaque of individual deciduous molars in 143 subjects. PK activity was measured at different time points during biofilm formation. Patients were divided into two groups: initial stage decay, and extensive decay. Non-parametric analysis of variance and analysis of covariance were used to determine the connections between S. mutans levels, PK activity and dental caries biotypes. A total of 143 strains were derived from subjects with caries. Biotyping data showed that 62, 23, 50, and 8 strains were classified as biotypes I, II, III, IV, respectively. PK activity in biotypes I, II, and IV was significantly higher in comparison to that in biotype III. The correlation between the level of S. mutans in dental plaque and PK activity was both statistically significant (p < 0.05) and positive. The greater the level of S. mutans in the biofilm (colony count and total biomass), the higher the PK activity; similarly, a low bacterial count correlated with low PK activity.

MCPIP1 (Monocyte Chemotactic Protein-1 Induced Protein) is an important regulator of inflammation and cell apoptosis, but its role in UVA-induced stress response in the epidermis has never been studied. We have found that moderate apoptosis-inducing dose of UVA (27J/cm2) increases the level of MCPIP1 expression in HaCaT cells and normal human keratinocytes (NHEK) within 6-9h after the treatment. MCPIP1 upregulation was dependent on the induction of p38, but not p53, as demonstrated by using p38 inhibitor SB203580 and p53 inducer RG7388, respectively. This increase was also blocked by antioxidants (α-tocopherol and ascorbic acid), suggesting the involvement of MCPIP1 in UVA-induced oxidative stress response. Si-RNA-mediated down-regulation of MCPIP1 expression in HaCaT cells resulted in increased sensitivity to UVA-induced DNA damage and apoptosis. This was accompanied by decreased phosphorylation of p53 and p38 in MCPIP1-silenced cells following UVA irradiation. The activation of p38 in response to low doses of ultraviolet radiation was postulated to be protective for p53-inactive cells. Therefore, MCPIP1 may favor the survival of p53-defective HaCaT cells by sustaining the activation of p38. This creates a loop of mutual positive regulation between p38 and MCPIP1 protein in HaCaT cells, providing the protection against the consequences of UVA irradiation.

Objectives: The aim of this study was to evaluate the role of the antioxidant barrier in the saliva of children with caries, and its impact on the colonization of cariogenic bacteria. Methods: This is a cross-sectional study of 81 children aged 1–5 years. Antioxidant levels and salivary bacterial profiles were measured. Patients were divided into two groups as follows: initial stage decay, termed non-cavitated (1–2 in International Caries Detection and Assessment System (ICDAS)), and extensive decay, termed cavitated lesions (5–6 in ICDAS). The control group includes children without caries. Results: The linear regression model demonstrated that the GSH, GSSG, GSH/GSSG, and total antioxidant capacity levels are influenced (P

The importance of monocyte chemotactic protein-1-induced protein 1 (MCPIP1) in the negative regulation of inflammatory reactions has already been extensively studied. However, its role in cancer is not yet established. We studied MCPIP1 gene expression in primary human neuroblastomas and several neuroblastoma cell lines. Our results showed a lack of MCPIP1 expression in primary neuroblastoma tumors. Moreover, it was found that the low expression of the protein measured in human neuroblastoma cell lines might be important for neuroblastoma survival, since enforced MCPIP1 gene expression in human neuroblastoma BE(2)-C cells caused a significant decrease in neuroblastoma cell viability and proliferation.

Anterior segment dysgenesis (ASD) encompasses a spectrum of ocular disorders affecting the structures of the anterior eye chamber. Mutations in several genes, involved in eye development, are implicated in this disorder. ASD is often accompanied by diverse multisystemic symptoms and another genetic cause, such as variants in genes encoding collagen type IV. Thus, a wide spectrum of phenotypes and underlying genetic diversity make fast and proper diagnosis challenging. Here, we used AMELIE, an automatic text mining tool that enriches data with the most up-to-date information from literature, and wANNOVAR, which is based on well-documented databases and incorporates variant filtering strategy to identify genetic variants responsible for severely-manifested ASD in a newborn child. This strategy, applied to trio sequencing data in compliance with ACMG 2015 guidelines, helped us find two compound heterozygous variants of the B3GLCT gene, of which c.660+1G>A (rs80338851) was previously associated with the phenotype of Peters plus syndrome (PPS), while the second, NM_194318.3:c.755delC (p.T252fs), in exon 9 of the same gene was noted for the first time. PPS, a very rare subtype of ASD, is a glycosylation disorder, where the dysfunctional B3GLCT gene product, O-fucose-specific β-1,3-glucosyltransferase, is ineffective in providing a noncanonical quality control system for proper protein folding in cells. Our study expands the mutation spectrum of the B3GLCT gene related to PPS. We suggest that the implementation of automatic text mining tools in combination with careful variant filtering could help translate sequencing results into diagnosis, thus, considerably accelerating the diagnostic process and, thereby, improving patient management.

The protein p53, known as the “Guardian of the Genome”, plays an important role in maintaining DNA integrity, providing protection against cancer-promoting mutations. Dysfunction of p53 is observed in almost every cancer, with 50% of cases bearing loss-of-function mutations/deletions in the TP53 gene. In the remaining 50% of cases the overexpression of HDM2 (mouse double minute 2, human homolog) protein, which is a natural inhibitor of p53, is the most common way of keeping p53 inactive. Disruption of HDM2-p53 interaction with the use of HDM2 antagonists leads to the release of p53 and expression of its target genes, engaged in the induction of cell cycle arrest, DNA repair, senescence, and apoptosis. The induction of apoptosis, however, is restricted to only a handful of p53wt cells, and, generally, cancer cells treated with HDM2 antagonists are not efficiently eliminated. For this reason, HDM2 antagonists were tested in combinations with multiple other therapeutics in a search for synergy that would enhance the cancer eradication. This manuscript aims at reviewing the recent progress in developing strategies of combined cancer treatment with the use of HDM2 antagonists.

The present study describes a family with multiple endocrine neoplasia type 1 (MEN1) caused by a previously undescribed in-frame deletion c.1246_1248delGCC (Ala416del) in the MEN1 gene. Evidence for the pathogenic character of this mutation, which triggers an aggressive clinical outcome, is demonstrated. Aggregation analysis in the tested family was strongly suggestive of causality of the detected mutation. This was supported by the analysis of LOH (loss of heterozygosity) in tumor-derived DNA and by computational analysis of the functional and structural implications of the mutation. Different phenotypic characteristics were identified among family members, which is typical for MEN1. Additionally, an unexpected disease inheritance pattern was observed in this kindred, in which either all or none of the siblings of one branch inherited the disease.

The generalized inflammatory response leads to activation of hundreds of genes transcribed in an established sequence in specialized cells. Transcriptome analysis of human monocyte-derived cells stimulated with IL-1beta or with monocyte chemotactic protein-1 (MCP-1) has led to the identification of a new inflammation-related gene ZC3H12A encoding a chain of 599 amino acids corresponding to a 66-kDa protein. The protein, given a provisional name of MCPIP1 (monocyte chemotactic protein-induced protein-1), is expressed in several human and murine tissues such as bone marrow, spleen, heart and placenta. In in vivo studies, mice with inactivated MCPIP1-encoding gene showed growth retardation, lymphadenopathy, splenomegaly and enhanced inflammatory symptoms. Principal molecular features of MCPIP1 include a single zinc finger motif, an RNase-like PIN domain and ubiquitin-binding domain. Reports from independent laboratories suggest that MCPIP1 may function also as a deubiquitinase. Although MCPIP1 is regarded by some authors as a new transcription factor or cell differentiation factor modulating angiogenesis or adipogenesis, its principal function appears to be downregulation of inflammatory responses through at least two independent mechanisms: increased degradation of cytokine mRNAs and inhibition of LPS- and IL-1-induced NF-kappaB signaling pathway. The interference with NF-kappaB activation is highly complex and includes TRAF6 and TANK interaction with the ubiquitin-associated (UBA) domain of MCPIP1. Purified MCPIP1 protein was reported to degrade specific mRNA and cleave K48- and K63-linked polyubiquitin chains. Although some structural features and the mechanism of action of MCPIP1 are not fully explained yet, its importance in the regulation of inflammatory reactions has been firmly established.

Introduction Apart from the environmental risk factors for differentiated thyroid carcinoma (DTC), such as iodine deficiency and ionising radiation, it seems that there are also other, biological risk factors, for example, familial predisposition to thyroid disease. Objectives The aim of the study was to assess the occurrence of thyroid disease in the families of patients with DTC. Patients and methods A case-control study was conducted in a group of 232 patients with DTC and in 342 age- and sex-matched healthy subjects. Eighty patients were diagnosed with follicular thyroid carcinoma, 127 with papillary thyroid carcinoma, and 25 with oxyphilic thyroid carcinoma. The questionnaire included questions on the presence of thyroid diseases in first-degree relatives. The relative risk of DTC and the effect of factors associated with thyroid diseases in the family were assessed by the logistic regression model. Results Thyroid disease was more common in the families of DTC patients than in the control group: 18.5% of the patients and 9.6% of the control group had a parent with thyroid disease (OR = 2.12, 95% CI: 1.26-3.55); 16.8% of the patients and 7.7% of the control group had a sibling with thyroid disease (OR = 2.27, 95% CI: 1.31-3.95). Conclusions Familial thyroid disease may be a risk factor for DTC. A positive family history of thyroid disease is associated to a larger extent with the development of papillary thyroid carcinoma than with that of follicular thyroid carcinoma.

Introduction Programmed cell death-1 (PD-1, CD279) protein is a surface receptor belonging to immunoglobulin superfamily. The receptor is expressed mostly on activated T cells and serves as an emergency brake for turning off the destructive action of these cells. Its natural ligand, PD-L1, is expressed on several types of immune cells to prevent from autoimmune reactions and provide T cell homeostasis. Due to its immunosuppressive function, PD-L1 is also utilised by a variety of cancer types to evade the deadly impact of activated T cells. In the recent years cancer immunotherapy utilising antibodies targeting PD-1/PD-L1 interaction has been proved to be exceptionally effective in multiple clinical studies. Due to extraordinary clinical outcome the use of both anti-PD-1 and anti-PD-L1 antibodies has been approved for the treatment of several cancer types. Despite great achievements of the use of therapeutic antibodies, a strikingly less progress has been done in the field of small molecules targeting this interaction. In this report the comparison of the in vitro activity of therapeutic antibodies, Bristol-Myers Squibb (BMS) small molecules and cyclic peptides is presented. Material and methods For the comparison of in vitro potential in blocking PD-1/PD-L1 axis, the PD-1/PD-L1 immune checkpoint blockade (ICB) assay (Promega) was utilised. Four therapeutic antibodies, i.e. nivolumab (Opdivo), pembrolizumab (Keytruda), atezolizumab (Tecentriq) and durvalumab (Imfinzi), two BMS small molecules (BMS-1001 and BMS-1166) and two BMS peptides (peptide-57 and peptide-71) were tested. Results and discussions For all the molecules tested the EC50 values of the immune checkpoint blocking activity were in sub-micromolar range. The experiments revealed the strongest ICB potential of the therapeutic antibodies, followed by cyclic peptides. The lowest activity was observed in the case of BMS small molecules. Conclusion Although the small molecules and cyclic peptides show the potential of blocking PD-1/PD-L1 interaction in vitro, the activity of therapeutic antibodies is still unrivalled. The optimisation of the structures of the compounds is required before proceeding to clinical trials.