Metallothionein-3 is a multifunctional driver that modulates the development of sorafenib-resistant phenotype in hepatocellular carcinoma cells.

BACKGROUND & AIMS: Metallothionein-3 (hMT3) is a structurally unique member of the metallothioneins family of low-mass cysteine-rich proteins. hMT3 has poorly characterized functions, and its importance for hepatocellular carcinoma (HCC) cells has not yet been elucidated. Therefore, we investigated the molecular mechanisms driven by hMT3 with a special emphasis on susceptibility to sorafenib. METHODS: Intrinsically sorafenib-resistant (BCLC-3) and sensitive (Huh7) cells with or without up-regulated hMT3 were examined using cDNA microarray and methods aimed at mitochondrial flux, oxidative status, cell death, and cell cycle. In addition, in ovo/ex ovo chick chorioallantoic membrane (CAM) assays were conducted to determine a role of hMT3 in resistance to sorafenib and associated cancer hallmarks, such as angiogenesis and metastastic spread. Molecular aspects of hMT3-mediated induction of sorafenib-resistant phenotype were delineated using mass-spectrometry-based proteomics. RESULTS: The phenotype of sensitive HCC cells can be remodeled into sorafenib-resistant one via up-regulation of hMT3. hMT3 has a profound effect on mitochondrial respiration, glycolysis, and redox homeostasis. Proteomic analyses revealed a number of hMT3-affected biological pathways, including exocytosis, glycolysis, apoptosis, angiogenesis, and cellular stress, which drive resistance to sorafenib. CONCLUSIONS: hMT3 acts as a multifunctional driver capable of inducing sorafenib-resistant phenotype of HCC cells. Our data suggest that hMT3 and related pathways could serve as possible druggable targets to improve therapeutic outcomes in patients with sorafenib-resistant HCC.

Engineered human H-chain ferritin with reversed charge of the internal cavity exhibits RNA-mediated spongelike effect for loading RNA/DNA-binding molecules.

Ferritins are globular proteins with an internal cavity that enables the encapsulation of a plethora of low-mass compounds. Unfortunately, the overall negative surface charge of ferritin's internal cavity hampers efficient loading of negatively charged molecules. Therefore, we produced a genetically engineered human H-chain ferritin containing a cationic RKRK domain, reversing the natural net charge of the cavity to positive, thus allowing for efficient encapsulation of negatively charged siRNA. Due to the reversed, positive charge mediated by RKRK domains, the recombinant ferritin produced in E. coli inherently carries a load of bacterial RNA inside its cavity, turning the protein into an effective sponge possessing high affinity for DNA/RNA-binding substances that can be loaded with markedly higher efficiency compared to the wildtype protein. Using doxorubicin as payload, we show that due to its loading through the RNA sponge, doxorubicin is released in a sustained manner, with a cytotoxicity profile similar to the free drug. In summary, this is the first report demonstrating a ferritin/nucleic acid hybrid delivery vehicle with a broad spectrum of properties exploitable in various fields of biomedical applications.

Solvent Accessibility Promotes Rotamer Errors during Protein Modeling with Major Side-Chain Prediction Programs.

Side-chain rotamer prediction is one of the most critical late stages in protein 3D structure building. Highly advanced and specialized algorithms (e.g., FASPR, RASP, SCWRL4, and SCWRL4v) optimize this process by use of rotamer libraries, combinatorial searches, and scoring functions. We seek to identify the sources of key rotamer errors as a basis for correcting and improving the accuracy of protein modeling going forward. In order to evaluate the aforementioned programs, we process 2496 high-quality single-chained all-atom filtered 30% homology protein 3D structures and use discretized rotamer analysis to compare original with calculated structures. Among 513,024 filtered residue records, increased amino acid residue-dependent rotamer errors─associated in particular with polar and charged amino acid residues (ARG, LYS, and GLN)─clearly correlate with increased amino acid residue solvent accessibility and an increased residue tendency toward the adoption of non-canonical off rotamers which modeling programs struggle to predict accurately. Understanding the impact of solvent accessibility now appears key to improved side-chain prediction accuracies.

Titanium dioxide nanoparticles: Recent progress in antimicrobial applications.

For decades, the antimicrobial applications of nanoparticles (NPs) have attracted the attention of scientists as a strategy for controlling the ever-increasing threat of multidrug-resistant microorganisms. The photo-induced antimicrobial properties of titanium dioxide (TiO2 ) NPs by ultraviolet (UV) light are well known. This review elaborates on the modern methods and antimicrobial mechanisms of TiO2 NPs and their modifications to better understand and utilize their potential in various biomedical applications. Additional compounds can be grafted onto TiO2 nanomaterial, leading to hybrid metallic or non-metallic materials. To improve the antimicrobial properties, many approaches involving TiO2 have been tested. The results of selected studies from the past few years covering the most recent trends in this field are discussed in this review. There is extensive evidence to show that TiO2 NPs can exhibit certain antimicrobial features with disputable roles of UV light. Hence, they are effective in treating bacterial infections, although the majority of these conclusions came from in vitro studies and in the presence of some additional nanomaterials. The methods of evaluation varied depending on the nature of the research while researchers incorporated different techniques, including determining the minimum inhibitory concentration, cell count, and using disk and well diffusion methods, with a noticeable indication that cell count was the most and dominant criterion used to evaluate the antimicrobial activity. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease.

Correction: The Zinc-Schiff Base-Novicidin Complex as a Potential Prostate Cancer Therapy.

[This corrects the article DOI: 10.1371/journal.pone.0163983.].

A rotamer relay information system in the epidermal growth factor receptor-drug complexes reveals clues to new paradigm in protein conformational change.

Cancer cells can escape the effects of chemotherapy through mutations and upregulation of a tyrosine kinase protein called the epidermal growth factor receptor (EGFR). In the past two decades, four generations of tyrosine kinase inhibitors targeting EGFR have been developed. Using comparative structure analysis of 116 EGFR-drug complex crystal structures, cluster analysis produces two clans of 73 and 43 structures, respectively. The first clan of 73 structures is larger and is comprised mostly of the C-helix-IN conformation while the second clan of 43 structures correlates with the C-helix-OUT conformation. A deep rotamer analysis identifies 43 residues (18%) of the total of 237 residues spanning the kinase structures under investigation with significant rotamer variations between the C-helix-IN and C-helix-OUT clans. The locations of these rotamer variations take on the appearance of side chain conformational relays extending out from points of EGFR mutation to different regions of the EGFR kinase. Accordingly, we propose that key EGFR mutations act singly or together to induce drug resistant conformational changes in EGFR that are communicated via these side chain conformational relays. Accordingly, these side chain conformational relays appear to play a significant role in the development of tumour resistance. This phenomenon also suggests a new paradigm in protein conformational change that is mediated by supportive relays of rotamers on the protein surface, rather than through conventional backbone movements.

Metallothionein-3 promotes cisplatin chemoresistance remodelling in neuroblastoma.

Metallothionein-3 has poorly characterized functions in neuroblastoma. Cisplatin-based chemotherapy is a major regimen to treat neuroblastoma, but its clinical efficacy is limited by chemoresistance. We investigated the impact of human metallothionein-3 (hMT3) up-regulation in neuroblastoma cells and the mechanisms underlying the cisplatin-resistance. We confirmed the cisplatin-metallothionein complex formation using mass spectrometry. Overexpression of hMT3 decreased the sensitivity of neuroblastoma UKF-NB-4 cells to cisplatin. We report, for the first time, cisplatin-sensitive human UKF-NB-4 cells remodelled into cisplatin-resistant cells via high and constitutive hMT3 expression in an in vivo model using chick chorioallantoic membrane assay. Comparative proteomic analysis demonstrated that several biological pathways related to apoptosis, transport, proteasome, and cellular stress were involved in cisplatin-resistance in hMT3 overexpressing UKF-NB-4 cells. Overall, our data confirmed that up-regulation of hMT3 positively correlated with increased cisplatin-chemoresistance in neuroblastoma, and a high level of hMT3 could be one of the causes of frequent tumour relapses.

Metallothionein dimerization evidenced by QD-based Förster resonance energy transfer and capillary electrophoresis.

Herein, we report a new simple and easy-to-use approach for the characterization of protein oligomerization based on fluorescence resonance energy transfer (FRET) and capillary electrophoresis with LED-induced detection. The FRET pair consisted of quantum dots (QDs) used as an emission tunable donor (emission wavelength of 450 nm) and a cyanine dye (Cy3), providing optimal optical properties as an acceptor. Nonoxidative dimerization of mammalian metallothionein (MT) was investigated using the donor and acceptor covalently conjugated to MT. The main functions of MTs within an organism include the transport and storage of essential metal ions and detoxification of toxic ions. Upon storage under aerobic conditions, MTs form dimers (as well as higher oligomers), which may play an essential role as mediators in oxidoreduction signaling pathways. Due to metal bridging by Cd2+ ions between molecules of metallothionein, the QDs and Cy3 were close enough, enabling a FRET signal. The FRET efficiency was calculated to be in the range of 11-77%. The formation of MT dimers in the presence of Cd2+ ions was confirmed by MALDI-MS analyses. Finally, the process of oligomerization resulting in FRET was monitored by CE, and oligomerization of MT was confirmed.

Toward structure-based drug design against the epidermal growth factor receptor (EGFR).

Most of the available crystal structures of epidermal growth factor receptor (EGFR) kinase domain, bound to drug inhibitors, originated from ligand-based drug design studies. Here, we used variations in 110 crystal structures to assemble eight distinct families highlighting the C-helix orientation in the N-lobe of the EGFR kinase domain. The families shared similar mutational profiles and similarity in the ligand R-groups (chemical composition, geometry, and charge) facing the C-helix, mutation sites, and DFG domain. For structure-based drug design, we recommend a systematic decision-making process for choice of template, guided by appropriate pairwise fitting and clustering before the molecular docking step. Alternatively, the binding site shape/volume can be used to filter and select the compound libraries.

Homology modeling in the time of collective and artificial intelligence.

Homology modeling is a method for building protein 3D structures using protein primary sequence and utilizing prior knowledge gained from structural similarities with other proteins. The homology modeling process is done in sequential steps where sequence/structure alignment is optimized, then a backbone is built and later, side-chains are added. Once the low-homology loops are modeled, the whole 3D structure is optimized and validated. In the past three decades, a few collective and collaborative initiatives allowed for continuous progress in both homology and ab initio modeling. Critical Assessment of protein Structure Prediction (CASP) is a worldwide community experiment that has historically recorded the progress in this field. Folding@Home and Rosetta@Home are examples of crowd-sourcing initiatives where the community is sharing computational resources, whereas RosettaCommons is an example of an initiative where a community is sharing a codebase for the development of computational algorithms. Foldit is another initiative where participants compete with each other in a protein folding video game to predict 3D structure. In the past few years, contact maps deep machine learning was introduced to the 3D structure prediction process, adding more information and increasing the accuracy of models significantly. In this review, we will take the reader in a journey of exploration from the beginnings to the most recent turnabouts, which have revolutionized the field of homology modeling. Moreover, we discuss the new trends emerging in this rapidly growing field.

Characterization and in vitro Analysis of Probiotic-Derived Peptides Against Multi Drug Resistance Bacterial Infections.

An inexorable switch from antibiotics has become a major desideratum to overcome antibiotic resistance. Bacteriocin from Lactobacillus casei, a cardinal probiotic was used to design novel antibacterial peptides named as Probiotic Bacteriocin Derived and Modified (PBDM) peptides (PBDM1: YKWFAHLIKGLC and PBDM2: YKWFRHLIKKLC). The loop-shaped 3D structure of peptides was characterized in silico via molecular dynamics simulation as well as biophysically via spectroscopic methods. Thereafter, in vitro results against multidrug resistant bacterial strains and hospital samples demonstrated the strong antimicrobial activity of PBDM peptides. Further, in vivo studies with PBDM peptides showed downright recovery of balb/c mice from Vancomycin Resistant Staphylococcus aureus (VRSA) infection to its healthy condition. Thereafter, in vitro study with human epithelial cells showed no significant cytotoxic effects with high biocompatibility and good hemocompatibility. In conclusion, PBDM peptides displayed significant antibacterial activity against certain drug resistant bacteria which cause infections in human beings. Future analysis are required to unveil its mechanism of action in order to execute it as an alternative to antibiotics.

Norepinephrine transporter-derived homing peptides enable rapid endocytosis of drug delivery nanovehicles into neuroblastoma cells.

BACKGROUND: Currently, the diagnosis and treatment of neuroblastomas-the most frequent solid tumors in children-exploit the norepinephrine transporter (hNET) via radiolabeled norepinephrine analogs. We aim to develop a nanomedicine-based strategy towards precision therapy by targeting hNET cell-surface protein with hNET-derived homing peptides. RESULTS: The peptides (seq. GASNGINAYL and SLWERLAYGI) were shown to bind high-resolution homology models of hNET in silico. In particular, one unique binding site has marked the sequence and structural similarities of both peptides, while most of the contribution to the interaction was attributed to the electrostatic energy of Asn and Arg (< - 228 kJ/mol). The peptides were comprehensively characterized by computational and spectroscopic methods showing ~ 21% ß-sheets/aggregation for GASNGINAYL and ~ 27% α-helix for SLWERLAYGI. After decorating 12-nm ferritin-based nanovehicles with cysteinated peptides, both peptides exhibited high potential for use in actively targeted neuroblastoma nanotherapy with exceptional in vitro biocompatibility and stability, showing minor yet distinct influences of the peptides on the global expression profiles. Upon binding to hNET with fast binding kinetics, GASNGINAYLC peptides enabled rapid endocytosis of ferritins into neuroblastoma cells, leading to apoptosis due to increased selective cytotoxicity of transported payload ellipticine. Peptide-coated nanovehicles significantly showed higher levels of early apoptosis after 6 h than non-coated nanovehicles (11% and 7.3%, respectively). Furthermore, targeting with the GASNGINAYLC peptide led to significantly higher degree of late apoptosis compared to the SLWERLAYGIC peptide (9.3% and 4.4%, respectively). These findings were supported by increased formation of reactive oxygen species, down-regulation of survivin and Bcl-2 and up-regulated p53. CONCLUSION: This novel homing nanovehicle employing GASNGINAYLC peptide was shown to induce rapid endocytosis of ellipticine-loaded ferritins into neuroblastoma cells in selective fashion and with successful payload. Future homing peptide development via lead optimization and functional analysis can pave the way towards efficient peptide-based active delivery of nanomedicines to neuroblastoma cells.

Metallothionein isoforms as double agents - Their roles in carcinogenesis, cancer progression and chemoresistance.

Metallothioneins (MTs) are small cysteine-rich intracellular proteins with four major isoforms identified in mammals, designated MT-1 through MT-4. The best known biological functions of MTs are their ability to bind and sequester metal ions as well as their active role in redox homeostasis. Despite these protective roles, numerous studies have demonstrated that changes in MT expression could be associated with the process of carcinogenesis and participation in cell differentiation, proliferation, migration, and angiogenesis. Hence, MTs have the role of double agents, i.e., working with and against cancer. In view of their rich biochemical properties, it is not surprising that MTs participate in the emergence of chemoresistance in tumor cells. Many studies have demonstrated that MT overexpression is involved in the acquisition of resistance to anticancer drugs including cisplatin, anthracyclines, tyrosine kinase inhibitors and mitomycin. The evidence is gradually increasing for a cellular switch in MT functions, showing that they indeed have two faces: protector and saboteur. Initially, MTs display anti-oncogenic and protective roles; however, once the oncogenic process was launched, MTs are utilized by cancer cells for progression, survival, and contribution to chemoresistance. The duality of MTs can serve as a potential prognostic/diagnostic biomarker and can therefore pave the way towards the development of new cancer treatment strategies. Herein, we review and discuss MTs as tumor disease markers and describe their role in chemoresistance to distinct anticancer drugs.

Zinc phosphate-based nanoparticles as alternatives to zinc oxide in diet of weaned piglets.

BACKGROUND: The high doses of zinc oxide (ZnO) administered orally to piglets for the prevention of diarrhea and increase of growth rate can contaminate pig farms and the surrounding environment. Therefore, there is a need to find a replacement of high doses of dietary ZnO with an equally effective alternative. In the present study, the effect of two formulations of zinc phosphate-based nanoparticles (ZnA and ZnC NPs) on growth performance, intestinal microbiota, antioxidant status, and intestinal and liver morphology was evaluated. A total of 100 weaned piglets were randomly divided into 10 equal groups with the base diet (control) or the base diet supplemented with ZnA, ZnC, or ZnO at concentrations 500, 1000, and 2000 mg Zn per kilogram of diet. Supplements were given to animals for 10 days. Fecal samples were collected on day 0, 5, 10 and 20. At the end of the treatment (day 10), three piglets from each group were sacrificed and analyzed. RESULTS: Comparing to that of control, the significantly higher piglet weight gain was observed in all piglet groups fed with ZnA (P < 0.05). Differences in the total aerobic bacteria and coliform counts in piglet feces after NPs supplementation compared to that of control and ZnO groups were also found (P < 0.05). The majority of aerobic culturable bacteria from the feces represented Escherichia (28.57-47.62%), Enterococcus (3.85-35.71%), and Streptococcus (3.70-42.31%) spp. A total of 542 Escherichia coli isolates were screened for the virulence genes STa, STb, Stx2, F4, and F18. The substantial occurrence of E. coli virulence factors was found on day 5, mainly in fimbrillary antigen and thermostable toxins, except for piglets fed by ZnC. Zn treatment decreased Zn blood levels in piglets fed with ZnO and ZnA (500 mg/kg) and increased in ZnC (2000 mg/kg) compared to that of control (P < 0.05). The antioxidant status of piglets was affected only by ZnA. While some changes in the liver and the intestinal morphology of piglets with NPs were observed, none were serious as reflected by the normal health status and increased weigh gain performance. CONCLUSIONS: Our results indicate that ZnA NPs have a positive effect on the piglet growth performance even at the lowest concentration. The prevalence of E. coli virulence factors was lowest in pigs supplemented with ZnC. Zinc phosphate-based nanoparticles may be an effective alternative to ZnO.

LAT1 (SLC7A5) Overexpression in Negative Her2 Group of Breast Cancer: A Potential Therapy Target.

OBJECTIVE: HER2 negative carcinomas of the breast pose a challenge for treatment due to redundancies in potential drug targets and poor patient outcomes. Our aim was to investigate the role of L-type amino acid transporter - LAT1 as a potential prognosticator and a drug target. METHODS: In this retrospective work, we have studied the expression of LAT1 in 145 breast cancer tissues via immunohistochemistry. Overall survival analysis was used to evaluate patient outcome in various groups of our cohort. RESULTS: Positive LAT1 expression was found in 27 (84.4%) luminal A subtype, 27 (64.3%) luminal B/triple positive subtype, 29 (82.9%) triple negative subtype, and 24 (66.7%) HER2-only positive subtype (p=0.1). Interestingly, negative correlation was found between LAT1 and HER2; where positive expression of LAT1 was found in 56 (83.6%) cases in negative HER2 group and 51 (65.4%) cases from positive HER2 group (p=0.01). Unfortunately, we were unable to report significant survival differences when LAT1 expression was studied in the negative HER2 group. Nevertheless, five incidents of mortality (out of 55) were reported in LAT1+/HER2- group compared to none in the LAT1-/HER2- group (N=11). CONCLUSION: Our findings of overexpression of LAT1 in negative HER2 group suggest a role of this protein as prognosticator and drug target in a challenging therapeutic cohort.
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Novel mutations in the SMPD1 gene in Jordanian children with Acid sphingomyelinase deficiency (Niemann-Pick types A and B).

Acid sphingomyelinase (ASM) deficiency (ASMD) is a spectrum that includes Niemann-Pick disease (NPD) types A (NPD A) and B (NPD B). ASMD is characterized by intracellular accumulation of unesterified cholesterol and gangliosides within the endosomal-lysosomal system. It is caused by different mutations in SMPD1 gene that result in reduction or complete absence of acid sphingomyelinase activity in the cells. Herein, four unrelated consanguineous families with two NPD A and three NPD B patients were assessed for their genotypes via sequencing of the SMPD1 gene and their acid sphingomyelinase enzymatic activity. Among the eight identified mutations, three were novel and reported for the first time in Jordanian families (c.120_131delGCTGGCGCTGGC or c.132_143delGCTGGCGCTGGC, c.1758T > G, and c.1344T > A). All the patients displayed ASM activity lower than 1.3 µmol/l/h (P < 0.001). Genotyping and enzymatic assessment might play a significant role in disease identification in people at risk to facilitate genetic counseling in the future.

Ten quick tips for homology modeling of high-resolution protein 3D structures.

The purpose of this quick guide is to help new modelers who have little or no background in comparative modeling yet are keen to produce high-resolution protein 3D structures for their study by following systematic good modeling practices, using affordable personal computers or online computational resources. Through the available experimental 3D-structure repositories, the modeler should be able to access and use the atomic coordinates for building homology models. We also aim to provide the modeler with a rationale behind making a simple list of atomic coordinates suitable for computational analysis abiding to principles of physics (e.g., molecular mechanics). Keeping that objective in mind, these quick tips cover the process of homology modeling and some postmodeling computations such as molecular docking and molecular dynamics (MD). A brief section was left for modeling nonprotein molecules, and a short case study of homology modeling is discussed.

Peptide-Carbon Quantum Dots conjugate, Derived from Human Retinoic Acid Receptor Responder Protein 2, against Antibiotic-Resistant Gram Positive and Gram Negative Pathogenic Bacteria.

Antibiotic-resistant bacterial infections have become global issues for public health, which increases the utter need to develop alternatives to antibiotics. Here, the HSER (Homo sapiens retinoic acid receptor) peptide was designed from retinoic acid receptor responder protein 2 of Homo sapiens, and was conjugated with synthesized CQDs (carbon quantum dots) for enhanced antibacterial activity in combination, as individually they are not highly effective. The HSER-CQDs were characterized using spectrophotometer, HPLC coupled with electrospray-ionization quadrupole time-of-flight mass spectrometer (ESI-qTOF) mass spectrometer, zeta potential, zeta size, and FTIR. Thereafter, the antibacterial activity against Vancomycin-Resistant Staphylococcus aureus (VRSA) and Escherichia coli (carbapenem resistant) was studied using growth curve analysis, further supported by microscopic images showing the presence of cell debris and dead bacterial cells. The antibacterial mechanism of HSER-CQDs was observed to be via cell wall disruption and also interaction with gDNA (genomic DNA). Finally, toxicity test against normal human epithelial cells showed no toxicity, confirmed by microscopic analysis. Thus, the HSER-CQDs conjugate, having high stability and low toxicity with prominent antibacterial activity, can be used as a potential antibacterial agent.

Identification and Characterization of BTD Gene Mutations in Jordanian Children with Biotinidase Deficiency.

Biotinidase deficiency is an autosomal recessive metabolic disorder whose diagnosis currently depends on clinical symptoms and a biotinidase enzyme assay. This study aimed to investigate the mutational status and enzymatic activity of biotinidase deficiency in seven unrelated Jordanian families including 10 patients and 17 healthy family members. Amplified DNA was analyzed by the automated Sanger sequencing method, and the enzymatic assay was performed using a colorimetric assessment. Biotinidase level was significantly lower (p < 0.001) in BTD children compare to their non-affected family members. Genetic sequencing revealed six different mutations in Jordanian patients. One mutation was novel and located in exon 4, which could be a prevalent mutation for biotinidase deficiency in the Jordanian population. Identification of these common mutations and combing the enzymatic activity with genotypic data will help clinicians with regard to better genetic counseling and management through implementing prevention programs in the future.