Development of a bioluminescent homogenous nanobody-based immunoassay for the detection of prostate-specific antigen (PSA).

Prostate cancer is the most prevalent cancer in men. At present, the diagnosis and screening of prostate cancer rely on the essential biomarker known as prostate-specific antigen (PSA). The main purpose of this study was to develop a novel immunoassay for the detection of PSA based on a tri-part split-nanoluciferase system and a nanobody targeting PSA. In our approach, two small components of the split-nanoluciferase, referred to as ß9 and ß10, were individually fused to two anti-PSA nanobodies, N7 and N23. When these proteins bind to PSA and in the presence of the third nanoluciferase component, called Δ11S, the split-nanoluciferase components are brought into close proximity, facilitating the reassembly of the active nanoluciferase and activation of luminescence. These proteins were expressed in a bacterial expression system, purified, and employed for the intended immunoassay. The developed immunoassay demonstrated the capability to sensitively detect PSA within a linear range from 1.0 to 20.0 ng/mL with LOD of 0.4 ng/mL, and the results obtained through this immunoassay agreed with those derived from the ELISA. Our study indicates that the homogeneous immunoassay developed with nanobodies exhibits remarkable specificity for PSA and can serve as a reliable, fast, and user-friendly test for detecting PSA.

Gene biomarkers and classifiers for various subtypes of HTLV-1-caused ATLL cancer identified by a combination of differential gene co­expression and support vector machine algorithms.

Adult T-cell leukemia/lymphoma (ATLL) is pathogen-caused cancer that is progressed after the infection by human T-cell leukemia virus type 1. Four significant subtypes comprising acute, lymphoma, chronic, and smoldering have been identified for this cancer. However, there are no trustworthy prognostic biomarkers for these subtypes. We utilized a combination of two powerful network-based and machine-learning algorithms including differential co-expressed genes (DiffCoEx) and support vector machine-recursive feature elimination with cross-validation (SVM-RFECV) methods to categorize disparate ATLL subtypes from asymptomatic carriers (ACs). The results disclosed the significant involvement of CBX6, CNKSR1, and MAX in chronic, MYH10 and P2RY1 in acute, C22orf46 and HNRNPA0 in smoldering subtypes. These genes also can classify each ATLL subtype from AC carriers. The integration of the results of two powerful algorithms led to the identification of reliable gene classifiers and biomarkers for diverse ATLL subtypes.

Potential miRNA-gene interactions determining progression of various ATLL cancer subtypes after infection by HTLV-1 oncovirus.

BACKGROUND: Adult T-cell Leukemia/Lymphoma (ATLL) is a rapidly progressing type of T-cell non-Hodgkin lymphoma that is developed after the infection by human T-cell leukemia virus type 1 (HTLV-1). It could be categorized into four major subtypes, acute, lymphoma, chronic, and smoldering. These different subtypes have some shared clinical manifestations, and there are no trustworthy biomarkers for diagnosis of them. METHODS: We applied weighted-gene co-expression network analysis to find the potential gene and miRNA biomarkers for various ATLL subtypes. Afterward, we found reliable miRNA-gene interactions by identifying the experimentally validated-target genes of miRNAs. RESULTS: The outcomes disclosed the interactions of miR-29b-2-5p and miR-342-3p with LSAMP in ATLL_acute, miR-575 with UBN2, miR-342-3p with ZNF280B, and miR-342-5p with FOXRED2 in ATLL_chronic, miR-940 and miR-423-3p with C6orf141, miR-940 and miR-1225-3p with CDCP1, and miR-324-3p with COL14A1 in ATLL_smoldering. These miRNA-gene interactions determine the molecular factors involved in the pathogenesis of each ATLL subtype and the unique ones could be considered biomarkers. CONCLUSION: The above-mentioned miRNAs-genes interactions are suggested as diagnostic biomarkers for different ATLL subtypes.

A recombinant affitoxin derived from a HER3 affibody and diphteria-toxin has potent and selective antitumor activity.

High expression of receptor tyrosine-protein kinase erbB-3 (HER3) has been found in several malignancies such as breast cancer. In this study, we designed, produced and evaluated a new affitoxin consisting of a truncated form of diphtheria toxin and a HER3-binding affibody domains. The new affitoxin was expressed in Escherichia coli and purified by affinity chromatography. We evaluated the suitability of affitoxin to kill HER3 positive breast cancer cells with MTT and apoptosis assays. The protein synthesis inhibition was also evaluated. The IC50 value in HER3 negative cells is about 10 times more than HER3 positive cells in new design of affitoxin. The specificity of affitoxin for binding to HER3 positive cells was also investigated with binding assay with flow cytometry. The results show that, the new affitoxin is an anti-cancer molecule with specific binding to HER3 positive cells and may open a new window for the treatment of HER3-positive cancers.

Exploration of mRNAs and miRNA classifiers for various ATLL cancer subtypes using machine learning.

BACKGROUND: Adult T-cell Leukemia/Lymphoma (ATLL) is a cancer disease that is developed due to the infection by human T-cell leukemia virus type 1. It can be classified into four main subtypes including, acute, chronic, smoldering, and lymphoma. Despite the clinical manifestations, there are no reliable diagnostic biomarkers for the classification of these subtypes. METHODS: Herein, we employed a machine learning approach, namely, Support Vector Machine-Recursive Feature Elimination with Cross-Validation (SVM-RFECV) to classify the different ATLL subtypes from Asymptomatic Carriers (ACs). The expression values of multiple mRNAs and miRNAs were used as the features. Afterward, the reliable miRNA-mRNA interactions for each subtype were identified through exploring the experimentally validated-target genes of miRNAs. RESULTS: The results revealed that miR-21 and its interactions with DAAM1 and E2F2 in acute, SMAD7 in chronic, MYEF2 and PARP1 in smoldering subtypes could significantly classify the diverse subtypes. CONCLUSIONS: Considering the high accuracy of the constructed model, the identified mRNAs and miRNA are proposed as the potential therapeutic targets and the prognostic biomarkers for various ATLL subtypes.

Integration of gene co-expression analysis and multi-class SVM specifies the functional players involved in determining the fate of HTLV-1 infection toward the development of cancer (ATLL) or neurological disorder (HAM/TSP).

Human T-cell Leukemia Virus type-1 (HTLV-1) is an oncovirus that may cause two main life-threatening diseases including a cancer type named Adult T-cell Leukemia/Lymphoma (ATLL) and a neurological and immune disturbance known as HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). However, a large number of the infected subjects remain as asymptomatic carriers (ACs). There is no comprehensive study that determines which dysregulated genes differentiate the pathogenesis routes toward ATLL or HAM/TSP. Therefore, two main algorithms including weighted gene co-expression analysis (WGCNA) and multi-class support vector machines (SVM) were utilized to find major gene players in each condition. WGCNA was used to find the highly co-regulated genes and multi-class SVM was employed to identify the most important classifier genes. The identified modules from WGCNA were validated in the external datasets. Furthermore, to find specific modules for ATLL and HAM/TSP, the non-preserved modules in another condition were found. In the next step, a model was constructed by multi-class SVM. The results revealed 467, 3249, and 716 classifiers for ACs, ATLL, and HAM/TSP, respectively. Eventually, the common genes between the WGCNA results and classifier genes resulted from multi-class SVM that also determined as differentially expressed genes, were identified. Through these step-wise analyses, PAIP1, BCAS2, COPS2, CTNNB1, FASLG, GTPBP1, HNRNPA1, RBBP6, TOP1, SLC9A1, JMY, PABPC3, and PBX1 were found as the possible critical genes involved in the progression of ATLL. Moreover, FBXO9, ZNF526, ERCC8, WDR5, and XRCC3 were identified as the conceivable major involved genes in the development of HAM/TSP. These genes can be proposed as specific biomarker candidates and therapeutic targets for each disease.

Bioprocess optimization of interferon ß-1-a in Pichia pastoris and its improved inhibitory effect against hepatocellular carcinoma cells

Interferon-ß-1a (INF-ß-1a) has gained significant attention due to its emerging applications in the treatment of different human diseases. Therefore, many researchers have attempted to produce it in large quantities and also in a biologically active form using different expression systems. In the present study, we aimed to improve the expression level of INF-ß-1a by Pichia pastoris using optimization of culture conditions. The codon-optimized INF-ß- 1a gene was cloned into pPICZαA plasmid under the control of alcohol oxidase I (AOX1) promoter. The protein expression was induced using different concentrations of methanol at different pHs and temperatures. The biological activity of produced protein was evaluated by anti-proliferative assay. The ideal culture conditions for the expression of INF-ß-1a by P. pastoris were found to be induction with 2% methanol at pH 7.0 culture medium at 30 C which yielded a concentration of 15.5 mg/L INF-ß-1a in a shake flask. Our results indicate that differences in glycosylation pattern could result in different biological activities as INF- ß-1a produced by P. pastoris could significantly more reduce the cell viability of HepG-2 cells, a hepatocellular carcinoma cell line, than a commercially available form of this protein produced by CHO

Development of recombinant biomimetic nano-carrier for targeted gene transfer to HER3 positive breast cancer.

Human epidermal growth factor receptor 3 (HER3) has rapidly gained much attention as a promising target for cancer treatment. The increasing recognition of HER3 roles in a number of HER family-driven cancers has led to studies aimed at targeting this receptor and developing HER3-targeted platforms with the ability to deliver therapeutic genes. We have previously indicated that the flexible linker and one unit of RALA in affibody-based platform could target HER3 and deliver its cargo. Based on the previous finding, in a new class of affibody-based platforms, we used two different linkers and RALA units and then compared their effectiveness on targeting and delivering specified genes to HER3 positive cells. Our results clearly showed that our biopolymeric platforms can successfully condense DNA into nanoparticles and object the overexpressed HER3 receptors and then transfer specific genes. Our affibody-based platform containing a rigid linker and one RALA unit presents an adequate transfection efficacy and low toxicity (based on MTT and apoptosis assays), however, the platform containing two RALA units and a flexible linker demonstrated high transfection efficacy while having modest toxicity in HER3 positive breast cancer cells. This may pave the way for further innovative applications of recombinant biopolymer when stable and economical productions need to be definitely considered.

Decoding pathogenesis factors involved in the progression of ATLL or HAM/TSP after infection by HTLV-1 through a systems virology study.

BACKGROUND: Human T-cell Leukemia Virus type-1 (HTLV-1) is a retrovirus that causes two diseases including Adult T-cell Leukemia/Lymphoma (ATLL cancer) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP, a neurodegenerative disease) after a long latency period as an asymptomatic carrier (AC). There are no obvious explanations about how each of the mentioned diseases develops in the AC carriers. Finding the discriminative molecular factors and pathways may clarify the destiny of the infection. METHODS: To shed light on the involved molecular players and activated pathways in each state, differentially co-expressed modules (DiffCoEx) algorithm was employed to identify the highly correlated genes which were co-expressed differently between normal and ACs, ACs and ATLL, as well as ACs and HAM/TSP samples. Through differential pathway analysis, the dysregulated pathways and the specific disease-genes-pathways were figured out. Moreover, the common genes between the member of DiffCoEx and differentially expressed genes were found and the specific genes in ATLL and HAM/TSP were introduced as possible biomarkers. RESULTS: The dysregulated genes in the ATLL were mostly enriched in immune and cancer-related pathways while the ones in the HAM/TSP were enriched in immune, inflammation, and neurological pathways. The differential pathway analysis clarified the differences between the gene players in the common activated pathways. Eventually, the final analysis revealed the involvement of specific dysregulated genes including KIRREL2, RAB36, and KANK1 in HAM/TSP as well as LTB4R2, HCN4, FZD9, GRIK5, CREB3L4, TACR2, FRMD1, LHB, FGF3, TEAD3, GRIN2D, GNRH2, PRLH, GPR156, and CRHR2 in ATLL. CONCLUSION: The identified potential prognostic biomarkers and therapeutic targets are proposed as the most important platers in developing ATLL or HAM/TSP. Moreover, the proposed signaling network clarifies the differences between the functional players in the activated pathways in ACs, ATLL, and HAM/TSP.

Deciphering microRNA-mRNA regulatory network in adult T-cell leukemia/lymphoma; the battle between oncogenes and anti-oncogenes.

Adult T-cell leukemia/lymphoma (ATLL) is virus-caused cancer that originates from the infection by human T-cell leukemia virus type 1. ATLL dysregulates various biological pathways related to the viral infection and cancer progression through the dysexpression of miRNAs and mRNAs. In this study, the potential regulatory subnetworks were constructed aiming to shed light on the pathogenesis mechanism of ATLL. For this purpose, two mRNA and one miRNA expression datasets were firstly downloaded from the GEO database. Next, the differentially expressed genes and miRNAs (DEGs and DE-miRNAs, respectively), as well as differentially co-expressed gene pairs (DCGs), were determined. Afterward, common DEGs and DCGs targeted by experimentally validated DE-miRNAs were explored. The oncogenic and anti-oncogenic miRNA-mRNA regulatory subnetworks were then generated. The expression levels of four genes and two miRNAs were examined in the blood samples by qRT-PCR. The members of three oncogenic/anti-oncogenic subnetworks were generally enriched in immune, virus, and cancer-related pathways. Among them, FZD6, THBS4, SIRT1, CPNE3, miR-142-3p, and miR-451a were further validated by real-time PCR. The significant up-regulation of FZD6, THBS4, and miR-451a as well as down-regulation of CPNE3, SIRT1, and miR-142-3p were found in ATLL samples than normal samples. The identified oncogenic/anti-oncogenic subnetworks are pieces of the pathogenesis puzzle of ATLL. The ultimate winner is probably an oncogenic network that determines the final fate of the disease. The identified genes and miRNAs are proposed as novel prognostic biomarkers for ATLL.

Development of a ZHER3-Affibody-Targeted Nano-Vector for Gene Delivery to HER3-Overexpressed Breast Cancer Cells.

Despite the initial successes of gene delivery applications, they faced on several intrinsic drawbacks including toxicity and immunogenicity. Therefore, alternative gene-delivery systems derived from recombinant peptides have emerged and is rapidly developing. Human epidermal growth factor receptor-3 (HER3) shows high activity in tumor resistance to anti-human epidermal growth factor receptor 2 (HER2) therapies. In this study, an affibody molecule against HER3 is conjugated to a biomimetic peptide RALA (an amphipathic and cationic peptide enriched with arginine) and the ability of the fusion vector for targeting HER3 and afterward delivering specific genes in breast cancer cells is evaluated. The results demonstrate that the biopolymeric platform, which contains an affibody-conjugated RALA peptide, can effectively condense DNA into nanoparticles and target the overexpressed HER3 receptors in breast cancer cells and transfer specific genes. The use of such a recombinant biopolymer may pave the way for the development of sensitive and effective diagnostic and treatment tool for breast cancer.

Detection of a prostate cancer cell line using a bioluminescent affiprobe: An attempt to develop a new molecular probe for ex vivo studies.

Molecular probes have become an important tool to decipher cell and molecular events, as well as contributing to clinical diagnosis. In this study, we report on a novel bioluminescence affiprobe and evaluate its functionality by monitoring HER2 expression in the prostate cancer cells. The proposed bioluminescence affiprobe consists of a binding domain, an HER2 specific affibody molecule and a bioluminescent domain, recombinant Renilla luciferase. The experiments indicate that bioluminescence affiprobe can serve as a reliable and user-friendly probe for the detection of human HER2 positive prostate cells as well as ex vivo based detection of HER2-positive human prostate cancer specimens using luminometeric-based assays.

Optimized protocol for soluble prokaryotic expression, purification and structural analysis of human placenta specific-1(PLAC1).

Placenta specific -1 (PLAC1) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLAC1 transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLAC1 in soluble form with high yield. This limitation also complicates the structural studies of PLAC1, which is important for prediction of its physiological roles. To address this issue, we employed an expression matrix consisting of two expression vectors, five different E. coli hosts and five solubilization conditions to optimize production of full and truncated forms of human PLAC1. The recombinant proteins were then characterized using an anti-PLAC1-specific antibody in Western blotting (WB) and enzyme linked immunosorbent assay (ELISA). Structure of full length protein was also investigated using circular dichroism (CD). We demonstrated the combination of Origami™ and pCold expression vector to yield substantial amount of soluble truncated PLAC1 without further need for solubilization step. Full length PLAC1, however, expressed mostly as inclusion bodies with higher yield in Origami™ and Rosetta2. Among solubilization buffers examined, buffer containing Urea 2 M, pH 12 was found to be more effective. Recombinant proteins exhibited excellent reactivity as detected by ELISA and WB. The secondary structure of full length PLAC1 was considered by CD spectroscopy. Taken together, we introduced here a simple, affordable and efficient expression system for soluble PLAC1 production.

Functional expression and purification of recombinant Hepcidin25 production in Escherichia coli using SUMO fusion technology.

Hepcidin25 is a small cysteine-rich peptide hormone known as a new class of antimicrobial peptides. The purpose of the present study was to express, purify and investigate the antibacterial properties of recombinant human hepcidin25 protein production in Escherichia coli. Human hepcidin25 gene was optimized and fused to a small ubiquitin-related modifier (SUMO) gene for higher expression. Then SUMO-hepcidin25 was cloned into the pET-32a (+) vector and expressed in E. coli Origami. The fusion protein with a molecular weight of approximately 35kDa was analyzed on SDS-PAGE gel. The highest expression was observed after 6h induction and the fusion protein consisted approximately 47% of the total cellular protein. The purified SUMO-hepcidin25 purity was determined to be higher than 95%, with a final yield of 3.9mgl-1 of media. The recombinant hepcidin25 showed antibacterial activity against both Gram negative (Klebsiella pneumonia) and Gram positive (Staphylococcus aureus and Bacillus cereus) bacteria with minimum inhibitory concentrations (MICs) of 150µgml-1, 18.7µg/ml-1 and 37.5µg/ml-1, respectively. These results indicated that thioredoxin and SUMO dual fusion system is an efficient production system for synthesis functional human hepcidin25.

Antioxidant activity and protective role on protein glycation of synthetic aminocoumarins

Background: Synthesized aminocoumarins are heterocyclic compounds possessing potential for the treatment of insulin-dependent diabetes mellitus with unexplored anti-glycative action. Results: In this study 4-aminocoumarin derivatives (4-ACDs) were evaluated in vitro for antiglycation (AG) activities by using the human serum albumin (HSA)/glucose system, for 8 weeks of incubation. The glycation and conformational alteration of HSA in the presence of the tested compounds were evaluated by Congo red assay, fluorescence and circular dichroism spectroscopy. The antioxidant (AO) capacity were also tested by four different assays including: DPPH (2,2'-diphenyl-1-picrylhydrazyl radical), ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulphonate) diammonium salt), FRAP (ferric reducing antioxidant power) and β-carotene-linoleic acid assay. The tested compounds showed AG and AO effects. The intensity of the accomplished AO potential is related to the type of the used assay. Significant alterations in the secondary (monitored by CD spectropolarimetry) and tertiary structure (assessed by spectrofluorimetry) of HSA upon glycation were mitigated by the 4-ACDs, suggesting their suppressive role in the late stage (post-Amadori) of the HSA glycation. Conclusions: By the analogues, in vitro ascertained AO and AG properties of 4-ACD may be recognized as rationale for their protective role against oxidative changes of proteins, thereby precluding diabetic complications in humans.

Renilla luciferase-labeled Annexin V: a new probe for detection of apoptotic cells.

The Ca(2+)-dependent binding of Annexin V to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this paper, we report a new class of Annexin V-based probes for apoptosis. Luciferase from Renilla reniformis (RLuc) was linked to Annexin V and expressed successfully in a soluble form in Escherichia coli BL21 (DE3). The new probe, Rluc/Annexin V, was purified and functionally assayed for detection of apoptosis in actinomycin D-induced apoptotic Jurkat cells. Moreover, the spontaneous apoptosis in neutrophils was shown using the new probe. The results indicate that Rluc/Annexin V can bind to the apoptotic cells, and the signal of Renilla luciferase can be detected by luminometric measurements. The availability of Rluc/Annexin V may be of potential commercial interest for improving current apoptosis assays.