Lack of NAD(P)+ transhydrogenase activity in patients with primary adrenal insufficiency due to NNT variants.

BACKGROUND: Pathogenic variants in the nicotinamide nucleotide transhydrogenase gene (NNT) are a rare cause of primary adrenal insufficiency (PAI), as well as functional impairment of the gonads. OBJECTIVE: Despite the description of different homozygous and compound heterozygous NNT variants in PAI patients, the extent to which the function and expression of the mature protein are compromised remains to be clarified. DESIGN: The activity and expression of mitochondrial NAD(P)+ transhydrogenase (NNT) were analyzed in blood samples obtained from patients diagnosed with PAI due to genetically confirmed variants of the NNT gene (n = 5), heterozygous carriers as their parents (n = 8), and healthy controls (n = 26). METHODS: NNT activity was assessed by a reverse reaction assay standardized for digitonin-permeabilized peripheral blood mononuclear cells (PBMCs). The enzymatic assay was validated in PBMC samples from a mouse model of NNT absence. Additionally, the PBMC samples were evaluated for NNT expression by western blotting and reverse transcription quantitative polymerase chain reaction and for mitochondrial oxygen consumption. RESULTS: NNT activity was undetectable (<4% of that of healthy controls) in PBMC samples from patients, independent of the pathogenic genetic variant. In patients' parents, NNT activity was approximately half that of the healthy controls. Mature NNT protein expression was lower in patients than in the control groups, while mRNA levels varied widely among genotypes. Moreover, pathogenic NNT variants did not impair mitochondrial bioenergetic function in PBMCs. CONCLUSIONS: The manifestation of PAI in NNT-mutated patients is associated with a complete lack of NNT activity. Evaluation of NNT activity can be useful to characterize disease-causing NNT variants.

Uniformly shaped harmonization combines human transcriptomic data from different platforms while retaining their biological properties and differential gene expression patterns.

Introduction: Co-normalization of RNA profiles obtained using different experimental platforms and protocols opens avenue for comprehensive comparison of relevant features like differentially expressed genes associated with disease. Currently, most of bioinformatic tools enable normalization in a flexible format that depends on the individual datasets under analysis. Thus, the output data of such normalizations will be poorly compatible with each other. Recently we proposed a new approach to gene expression data normalization termed Shambhala which returns harmonized data in a uniform shape, where every expression profile is transformed into a pre-defined universal format. We previously showed that following shambhalization of human RNA profiles, overall tissue-specific clustering features are strongly retained while platform-specific clustering is dramatically reduced. Methods: Here, we tested Shambhala performance in retention of fold-change gene expression features and other functional characteristics of gene clusters such as pathway activation levels and predicted cancer drug activity scores. Results: Using 6,793 cancer and 11,135 normal tissue gene expression profiles from the literature and experimental datasets, we applied twelve performance criteria for different versions of Shambhala and other methods of transcriptomic harmonization with flexible output data format. Such criteria dealt with the biological type classifiers, hierarchical clustering, correlation/regression properties, stability of drug efficiency scores, and data quality for using machine learning classifiers. Discussion: Shambhala-2 harmonizer demonstrated the best results with the close to 1 correlation and linear regression coefficients for the comparison of training vs validation datasets and more than two times lesser instability for calculation of drug efficiency scores compared to other methods.

Molecular Cardiotoxic Effects of Proteasome Inhibitors Carfilzomib and Ixazomib and Their Combination with Dexamethasone Involve Mitochondrial Dysregulation.

With the development and approval of new proteasome inhibitors, proteasome inhibition is increasingly recognized in cancer therapy. Besides successful anti-cancer effects in hematological cancers, side effects such as cardiotoxicity are limiting effective treatment. In this study, we used a cardiomyocyte model to investigate the molecular cardiotoxic mechanisms of carfilzomib (CFZ) and ixazomib (IXZ) alone or in combination with the immunomodulatory drug dexamethasone (DEX) which is frequently used in combination therapies in the clinic. According to our findings, CFZ showed a higher cytotoxic effect at lower concentrations than IXZ. DEX combination attenuated the cytotoxicity for both proteasome inhibitors. All drug treatments caused a marked increase in K48 ubiquitination. Both CFZ and IXZ caused an upregulation in cellular and endoplasmic reticulum stress protein (HSP90, HSP70, GRP94, and GRP78) levels and DEX combination attenuated the increased stress protein levels. Importantly, IXZ and IXZ-DEX treatments caused upregulation of mitochondria fission and fusion gene expression levels higher than caused by CFZ and CFZ-DEX combination. The IXZ-DEX combination reduced the levels of OXPHOS proteins (Complex II-V) more than the CFZ-DEX combination. Reduced mitochondrial membrane potential and ATP production were detected with all drug treatments in cardiomyocytes. Our findings suggest that the cardiotoxic effect of proteasome inhibitors may be due to their class effect and stress response and mitochondrial dysfunction may be involved in the cardiotoxicity process.

Next-Generation Grade and Survival Expression Biomarkers of Human Gliomas Based on Algorithmically Reconstructed Molecular Pathways.

In gliomas, expression of certain marker genes is strongly associated with survival and tumor type and often exceeds histological assessments. Using a human interactome model, we algorithmically reconstructed 7494 new-type molecular pathways that are centered each on an individual protein. Each single-gene expression and gene-centric pathway activation was tested as a survival and tumor grade biomarker in gliomas and their diagnostic subgroups (IDH mutant or wild type, IDH mutant with 1p/19q co-deletion, MGMT promoter methylated or unmethylated), including the three major molecular subtypes of glioblastoma (proneural, mesenchymal, classical). We used three datasets from The Cancer Genome Atlas and the Chinese Glioma Genome Atlas, which in total include 527 glioblastoma and 1097 low grade glioma profiles. We identified 2724 such gene and 2418 pathway survival biomarkers out of total 17,717 genes and 7494 pathways analyzed. We then assessed tumor grade and molecular subtype biomarkers and with the threshold of AUC > 0.7 identified 1322/982 gene biomarkers and 472/537 pathway biomarkers. This suggests roughly two times greater efficacy of the reconstructed pathway approach compared to gene biomarkers. Thus, we conclude that activation levels of algorithmically reconstructed gene-centric pathways are a potent class of new-generation diagnostic and prognostic biomarkers for gliomas.

Exploring the Anticancer Effects of Brominated Plastoquinone Analogs with Promising Cytotoxic Activity in MCF-7 Breast Cancer Cells via Cell Cycle Arrest and Oxidative Stress Induction.

Plastoquinone analogs are privileged structures among the known antiproliferative natural product-based compound families. Exploiting one of these analogs as a lead structure, we report the investigation of the brominated PQ analogs (BrPQ) in collaboration with the National Cancer Institute of Bethesda within the Developmental Therapeutics Program (DTP). These analogs exhibited growth inhibition in the micromolar range across leukemia, non-small cell lung cancer (EKVX, HOP-92, and NCI-H522), colon cancer (HCT-116, HOP-92), melanoma (LOX IMVI), and ovarian cancer (OVCAR-4) cell lines. One brominated PQ analog (BrPQ5) was selected for a full panel five-dose in vitro assay by the NCI's Development Therapeutic Program (DTP) division to determine GI50, TGI, and LC50 parameters. The brominated PQ analog (BrPQ5) displayed remarkable activity against most tested cell lines, with GI50 values ranging from 1.55 to 4.41 µM. The designed molecules (BrPQ analogs) obeyed drug-likeness rules, displayed a favorable predictive Absorption, Distribution, Metabolism, and Excretion (ADME) profile, and an in silico simulation predicted a possible BrPQ5 interaction with proteasome catalytic subunits. Furthermore, the in vitro cytotoxic activity of BrPQ5 was assessed, and IC50 values for U-251 glioma, MCF-7 and MDA-MB-231 breast cancers, DU145 prostate cancer, HCT-116 colon cancer, and VHF93 fibroblast cell lines were evaluated using an MTT assay. MCF-7 was the most affected cell line, and the effects of BrPQ5 on cell proliferation, cell cycle, oxidative stress, apoptosis/necrosis induction, and proteasome activity were further investigated in MCF-7 cells. The in vitro assay results showed that BrPQ5 caused cytotoxicity in MCF-7 breast cancer cells via cell cycle arrest and oxidative stress induction. However, BrPQ5 did not inhibit the catalytic activity of the proteasome. These results provide valuable insights for further discovery of novel antiproliferative agents.

The Role of Extracellular Vesicles Secreted From Thermal Stress-Induced Adipose-Derived Stem Cells on Bone Regeneration.

PURPOSE: Extracellular vesicles (EVs) are responsible for intercellular communication. Mesenchymal stem cell-derived vesicles have been shown to have similar properties as functional mesenchymal stem cells. The aim of this study was to compare the therapeutic benefit of EVs obtained from adipose tissue-derived stem cells (ADSC) on bone repair whereas using ß-tricalcium phosphate (ß-TCP) biomaterial as a carrier. MATERIALS AND METHOD: A single critical size bone defect of 8 mm in diameter was created on the right side of rat calvarium using a custom-made punch needle. Animals were randomly divided into 5 groups: group 1 (no treatment), group 2 (bone graft), group 3 (ß-TCP + ADSC), group 4 (ß-TCP + EV), group 5 (ß-TCP). Eight weeks later, animals were sacrificed and histologic and radiologic evaluation was performed. RESULTS: Semiquantitative histologic scoring demonstrated significantly higher bone regeneration scores for groups 2, 3, and 4 compared to group 1. Radiologic imaging showed significantly higher bone mineral density for groups 2, 3, and 5 compared to group 1. There were no significant differences between treatment groups in either histologic or radiologic scoring. CONCLUSIONS: Our data showed that EVs provided from thermally induced ADSCs did not show any significant difference in bone regeneration when compared to ADSCs themselves. Future studies should focus on determining the optimum amount and content of EV application since these vary significantly depending on the microenvironment.

Interactome analysis of Bag-1 isoforms reveals novel interaction partners in endoplasmic reticulum-associated degradation.

Bag-1 is a multifunctional protein that regulates Hsp70 chaperone activity, apoptosis, and proliferation. The three major Bag-1 isoforms have different subcellular localizations and partly non-overlapping functions. To identify the detailed interaction network of each isoform, we utilized mass spectrometry-based proteomics and found that interactomes of Bag-1 isoforms contained many common proteins, with variations in their abundances. Bag-1 interactomes were enriched with proteins involved in protein processing and degradation pathways. Novel interaction partners included VCP/p97; a transitional ER ATPase, Rad23B; a shuttling factor for ubiquitinated proteins, proteasome components, and ER-resident proteins, suggesting a role for Bag-1 also in ER-associated protein degradation (ERAD). Bag-1 pull-down from cells and tissues from breast cancer patients validated these interactions and showed cancer-related prominence. Using in silico predictions we detected hotspot residues of Bag-1. Mutations of these residues caused loss of binding to protein quality control elements and impaired proteasomal activity in MCF-7 cells. Following CD147 glycosylation pattern, we showed that Bag-1 downregulated VCP/p97-dependent ERAD. Overall, our data extends the interaction map of Bag-1, and broadens its role in protein homeostasis. Targeting the interaction surfaces revealed in this study might be an effective strategy in the treatment of cancer.

Investigation of 3D-Printed Polycaprolactone-/Polyvinylpyrrolidone-Based Constructs.

The aim of this study is to evaluate the mechanical and biological performance of cartilage-like constructs produced by 3D printing. During the investigation, poly(ε-caprolactone) (PCL) and polyvinylpyrrolidone (PVP) were used as a matrix polymer and low-molecular-weight chitosan (CS), hyaluronic acid (HA), and alginic acid sodium salt (SA) were integrated separately with the polymer matrix to fabricate the constructs. Thermal, mechanical, morphology, and chemical properties and swelling, degradation, and biocompatibility behaviors were evaluated in detail. With the addition of 3 fillers, the melting temperature of the matrix increased with the addition of fillers, and PCL/3wt.%PVP/1wt.%HA had the highest melting temperature value. Mechanical characterization results demonstrated that the printed PCL/3wt.%PVP/1wt.%CS displayed the highest compressive strength of around 9.51 MPa. The compressive strength difference between the PCL/3wt.%PVP and PCL/3wt.%PVP/1wt.%CS was 5.38 MPa. Biocompatibility properties of the constructs were tested by mitochondrial dehydrogenase activity, and in vitro studies showed that the PCL/3wt.%PVP/1wt.%HA composite construct had more cell viability than the other constructs by making use of the mesenchymal stem cell line.

Differential Interactome Based Drug Repositioning Unraveled Abacavir, Exemestane, Nortriptyline Hydrochloride, and Tolcapone as Potential Therapeutics for Colorectal Cancers.

There is a critical requirement for alternative strategies to provide the better treatment in colorectal cancer (CRC). Hence, our goal was to propose novel biomarkers as well as drug candidates for its treatment through differential interactome based drug repositioning. Differentially interacting proteins and their modules were identified, and their prognostic power were estimated through survival analyses. Drug repositioning was carried out for significant target proteins, and candidate drugs were analyzed via in silico molecular docking prior to in vitro cell viability assays in CRC cell lines. Six modules (mAPEX1, mCCT7, mHSD17B10, mMYC, mPSMB5, mRAN) were highlighted considering their prognostic performance. Drug repositioning resulted in eight drugs (abacavir, ribociclib, exemestane, voriconazole, nortriptyline hydrochloride, theophylline, bromocriptine mesylate, and tolcapone). Moreover, significant in vitro inhibition profiles were obtained in abacavir, nortriptyline hydrochloride, exemestane, tolcapone, and theophylline (positive control). Our findings may provide new and complementary strategies for the treatment of CRC.

Higher proteotoxic stress rather than mitochondrial damage is involved in higher neurotoxicity of bortezomib compared to carfilzomib.

Proteasome inhibitors have great success for their therapeutic potential against hematologic malignancies. First generation proteasome inhibitor bortezomib induced peripheral neuropathy is considered as a limiting factor in chemotherapy and its second-generation counterpart carfilzomib is associated with lower rates of neurotoxicity. The mitochondrial toxicity (mitotoxicity) hypothesis arises from studies with animal models of bortezomib induced peripheral neuropathy. However, molecular mechanisms are not fully elucidated and the role of mitotoxicity in bortezomib and carfilzomib induced neurotoxicity has not been investigated comparatively. Herein, we characterized the neurotoxic effects of bortezomib and carfilzomib at the molecular level in human neuronal cells using LC-MS/MS analysis, flow cytometry, RT-qPCR, confocal microscopy and western blotting. We showed that bortezomib and carfilzomib affected the human neuronal proteome differently, and bortezomib caused higher proteotoxic stress via protein oxidation, protein K48-ubiquitination, heat shock protein expression upregulation and reduction of mitochondria membrane potential. Bortezomib and carfilzomib did not affect the gene expression levels related to mitochondrial dynamics (optic atrophy 1; OPA1, mitofusin 1; MFN1, mitofusin 2; MFN2, fission 1; FIS1, dynamin-related protein 1; DRP1) and overall mitophagy rate whereas, PINK1/Parkin mediated mitophagy gene expressions were altered with both drugs. Bortezomib and carfilzomib caused downregulation of the contents of mitochondrial oxidative phosphorylation complexes, voltage-dependent anion channel 1 (VDAC1) and uncoupling protein 2 (UCP2) similarly. Our findings suggest that, both drugs induce mitotoxicity besides proteotoxic stress in human neuronal cells and the higher incidence of neurotoxicity with bortezomib than carfilzomib is not directly related to mitochondrial pathways.

HSP70 Inhibition Leads to the Activation of Proteasomal System under Mild Hyperthermia Conditions in Young and Senescent Fibroblasts.

Aging has been characterized with the accumulation of oxidized proteins, as a consequence of progressive decline in proteostasis capacity. Among others, proteasomal system is an efficient protein turnover complex to avoid aggregation of oxidized proteins. Heat shock protein 70 (HSP70) is another critical player that is involved in some key processes including the correct folding of misfolded proteins and targeting aggregated proteins to the proteasome for rapid degradation. The aim of this study was to determine the role of proteasomal system and heat shock proteins to maintain proteome balance during replicative senescence in mild hyperthermia conditions. Our results demonstrated that HSP40/70 machinery is induced by mild hyperthermia conditions independent from senescence conditions. Since HSP70 is largely responsible for the rapidly inducible cell protection following hyperthermia, the activation of "heat shock response" resulted in the elevation of HSP40/70 expressions as well as the proteasome activity. Interestingly, when HSP70 expression was inhibited, increased proteasomal activation was shown to be responsive to mild hyperthermia. Since HSP70 is involved in various stress-related pathways such as oxidative and endoplasmic reticulum stress, depletion of HSP70 expression may induce proteasomal degradation to maintain proteome balance of the cell. Thus, our data suggests that in mild heat stress conditions, molecular chaperone HSP70 plays an important role to avoid protein oxidation and aggregation; however, activities of proteasomal system are induced when HSP70 expression is depleted.

Cytotoxicity of Different Nano Composite Resins on Human Gingival and Periodontal Ligament Fibroblast Cell Lines: An In Vitro Study.

The aim of this study is to determine the cytotoxicity of three different nano composite resins (CRs) on human gingival fibroblast (hGF) and periodontal ligament fibroblast (hPDLF) cell lines. These CRs selected were nanohybrid organic monomer-based Admira Fusion (AF), nanohybrid Bis-(acryloyloxymethyl) tricyclo 5.2.1.0.sup.2,6 decane-based Charisma Topaz (CT), and supra nano filled resin-based Estelite Quick Sigma (EQS). MTT assay was performed to assess the cytotoxicity of CRs at 24 h and one week. AF and EQS applied on hGF cells at 24 h and one week demonstrated similar cytotoxic outcomes. Cytotoxicity of CT on hGF cells at one week was higher than 24 h (p = 0.04). Cytotoxicity of CT on hGF cells was higher at 24 h (p = 0.002) and one week (p = 0.009) compared to control. All composites showed higher cytotoxicity on hPDLF cells at one week than the 24 h (AF; p = 0.02, CT; p = 0.02, EQS; p = 0.04). AF and EQS demonstrated lower cytotoxicity on hPDLF cells than the control group at 24 h (AF; p = 0.01, EQS; p = 0.001). CT was found more cytotoxic on hPDLF cells than the control (p = 0.01) and EQS group (p = 0.008) at one week. The cytotoxicity of CRs on hGF and hPDLF cells vary, according to the type of composites, cell types, and exposure time.

Glioblastoma cell adhesion properties through bacterial cellulose nanocrystals in polycaprolactone/gelatin electrospun nanofibers.

Glioblastoma (GBM), the most common and extremely lethal type of brain tumor, is resistant to treatment and shows high recurrence rates. In the last decades, it is indicated that standard two-dimensional (2D) cell culture is inadequate to improve new therapeutic strategies and drug development. Hence, well-mimicked three-dimensional (3D) tumor platforms are needed to bridge the gap between in vitro and in vivo cancer models. In this study, bacterial cellulose nano-crystal (BCNC) containing polycaprolactone (PCL) /gelatin (Gel) nanofibrous composite scaffolds were successfully fabricated by electrospinning for mimicking the extracellular matrix of GBM tumor. The fiber diameters in the nanofibrous matrix were increased with an increased concentration of BCNC. Moreover, fiber morphology changed from the smooth formation to the beaded formation by increasing the concentration of the BCNC suspension. In-vitro biocompatibilities of nanofibrous scaffolds were tested with U251 MG glioblastoma cells and improved cell adhesion and proliferation was compared with PCL/Gel. PCL/Gel/BCNC were found suitable for enhancing axon growth and elongation supporting communication between tumor cells and the microenvironment, triggering the process of tumor recurrence. Based on these results, PCL/Gel/BCNC composite scaffolds are a good candidate for biomimetic GBM tumor platform.