Generation of anti-SN38 antibody for loading efficacy and therapeutic monitoring of SN38-containing therapeutics.

SN38, one of the most potent anti-tumor analogues of the camptothecins (CPTs), has limitations in its direct formulation as an anticancer agent due to its super toxicity and poor solubility in water and pharmaceutically approved solvents. However, it has garnered significant scientific interest as a payload in conjugated nanomedicine platforms (e.g., SN-38lip, NK012, SNB-101, and ADCs) to enhance their effectiveness and safety. The development of these platforms necessitates a convenient quantitative determination of SN38 in preclinical and clinical studies, a need that our study directly addresses, offering a practical solution to a pressing problem in cancer research and drug development. This study details the meticulous process of generating poly and monoclonal antibodies (pAb and mAb) against SN38 and their application to measure the SN38 in naked and conjugated forms of SN38-conjugated ADCs. For this purpose, two haptens of SN38 were synthesized by introducing the glycine or 4-amino-4-oxobutanyol(glycine) moiety as a conjugation functional group of the SN38. IR, NMR and mass spectrometric techniques confirmed the chemical modifications of the haptens. The haptens were then conjugated to each bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) protein. The SN38-KLH conjugates were meticulously examined for immunization and generation of pAb and mAb. The immunization efficiency, reactivity, binding affinity, specificity, and cross-reactivity of purified pAb and mAb against Irinotecan, a model for the emergence of an SN38 derivative in clinical settings, were evaluated using ELISA and western blotting (WB) techniques. Conjugation efficiency of the SN38 to the KLH was increased using 4-amino-4-oxobutanyol(glycine) moiety, as its immunization efficacy was more to generate pAb. Furthermore, only this hapten could immunized mice to generate mAb recognizing SN38 with nanomolar equilibrium affinity. Our recent findings strongly support the notion that the generated pAb employed in developing an ELISA effectively ascertains the presence of SN38 in SN38-conjugated ADC, with a test midpoint EC50 of 2.5 µg/mL. Our study's unique contribution to the field lies in the development of specific antibodies against SN38 for measuring it on ADC, a feat that has not been achieved before. These immunoassays can be readily applied to detect other SN38-conjugate therapeutic platforms, thereby enhancing their clinical knowledge translation. The affinity of both pAb and mAb also meets the acceptance criteria for quantifying SN38 in fluidic material, as well as in Therapeutic drug monitoring (TDM) studies, a crucial aspect of personalized medicine. The potential applications of the anti-SN38 antibodies extend to reducing SN38-induced systemic toxicity through an inverse targeting strategy, a novel approach that piques further interest in our findings.

Nanofabrication of chitosan-based dressing to treat the infected wounds: in vitro and in vivo evaluations.

Aim: Here, an innovative kind of antibacterial nanocomposite film is developed by incorporating graphene oxide and zinc oxide into chitosan matrix. Materials & methods: Our dressing was fabricated using the solution casting method. Fourier transform infrared spectra and TGA-DTG clearly confirmed the structure of film dressing. Results & conclusion: Our results showed the tensile strength and elongation at the break of the films were 20.1 ± 0.7 MPa and 36 ± 10%, respectively. Our fabricated film could absorb at least three-times the fluid of its dry weight while being biocompatible, antibacterial, non-irritant and non-allergic. In addition, it accelerated the healing process of infected wounds by regulating epithelium thickness and the number of inflammatory cells, thus it may be useful for direct application to damaged infected wounds.

In this study, an innovative kind of antibacterial nanocomposite film is developed by incorporating graphene oxide and zinc oxide into chitosan matrix. Our antibacterial wound dressing was fabricated using the solution casting method. Our fabricated film could absorb at least three-times the fluid of its dry weight while being biocompatible, antibacterial, non-irritant and non-allergic. In addition, our film accelerated the healing process of infected wounds by regulating epithelium thickness and the number of inflammatory cells. thus it may be useful for direct application to damaged infected wounds.

Biocatalytic synthesis of oxadiazole thioethers and evaluation of their antitumor activity.

A multicomponent enzyme-catalyzed process is suggested for the synthesis of a novel series of 1,3,4-oxadiazole thioether derivatives with yields ranging from 65 to 94%. Novozym 435, the immobilized form of Candida antarctica lipase B (CALB), was found to efficiently catalyze the reaction. The products were evaluated for antitumor activities against two cancer cell lines, HT-29 (human colorectal cancer cell) and HepG2 (human liver cancer cell), by MTT assays. Among them, two compounds exhibited higher antitumor activities, for both cell lines, compared to doxorubicin. In silico molecular docking and computational ADME analysis were performed to propose a mode of action for the anti-cancer activities and to predict drug-likeness, respectively.

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.

Metabolic profiling of seminal plasma from teratozoospermia patients.

Teratozoospermia is one of conditions that can cause male infertility. The mechanism of teratozoospermia remains unclear. The knowledge of the metabolites in human seminal plasma (HSP) is meaningful for the pathological study of teratozoospermia. Analysis of changed metabolites in HSP can help understand the cellular mechanism, find the novel biomarkers and subsequently design a diagnosis test. In this study, the analysis of samples performed by proton nuclear magnetic resonance spectroscopy (1H NMR spectroscopy) to identify the various metabolites, with the aim of finding metabolic profiles and biomarkers related to male infertility. Eighteen de-regulated metabolites were identified in fertile men compared to teratozoospermia patients. These changes illustrate the deficiencies in absorption or metabolism of these metabolites in teratozoospermia. Furthermore, metabolic profiling showed that it is not possible to classify teratozoospermia based on teratozoospermia index (TZI). To the best of our knowledge, this is the first metabolic profiling analysis of HSP described the metabolic features of teratozoospermia in a holistic view.

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.

Metabolomics of Male Infertility: A New Tool for Diagnostic Tests.

Infertility is a major health issue worldwide. Males and females contribute equally to this problem. Diagnostic semen analysis fails to identify 50% of male infertility disorders. In this regard, metabolomics as a new field of omics has been suggested to have the potential of solving and diagnosis of the male infertility problems. Metabo-lome has a history of around 20 years. However, there are only limited metabolomics studies carried out regarding male infertility. In this review, the current metabolomics researches that have been done in infertile men were reviewed. Based on our own results, using human seminal plasma for metabolomics studies is highly recommended to find potential biomarkers and developing diagnosis tests for detection of main deficiencies in infertile men.

Antibody-Drug Conjugates: Possibilities and Challenges.

The design of Antibody Drug Conjugates (ADCs) as efficient targeting agents for tumor cell is still in its infancy for clinical applications. This approach incorporates the antibody specificity and cell killing activity of chemically conjugated cytotoxic agents. Antibody in ADC structure acts as a targeting agent and a nanoscale carrier to deliver a therapeutic dose of cytotoxic cargo into desired tumor cells. Early ADCs encountered major obstacles including, low blood residency time, low penetration capacity to tumor microenvironment, low payload potency, immunogenicity, unusual off-target toxicity, drug resistance, and the lack of stable linkage in blood circulation. Although extensive studies have been conducted to overcome these issues, the ADCs based therapies are still far from having high-efficient clinical outcomes. This review outlines the key characteristics of ADCs including tumor marker, antibody, cytotoxic payload, and linkage strategy with a focus on technical improvement and some future trends in the pipeline.

Metabolic Fingerprinting of Seminal Plasma from Non-obstructive Azoospermia Patients: Positive Versus Negative Sperm Retrieval.

BACKGROUND: Non-obstructive azoospermia (NOA) occurs in approximately 10% of infertile men. Retrieval of the spermatozoa from the testicle of NOA patients is an invasive approach. Seminal plasma is an excellent source for exploring to find the biomarkers for presence of spermatozoa in testicular tissue. The present discovery phase study aimed to use metabolic fingerprinting to detect spermatogenesis from seminal plasma in NOA patients as a non-invasive method. METHODS: In this study, 20 men with NOA were identified based on histological analysis who had their first testicular biopsy in 2015 at Avicenna Fertility Center, Tehran, Iran. They were divided into two groups, a positive testicular sperm extraction (TESE(+)) and a negative testicular sperm extraction (TESE(-)). Seminal plasma of NOA patients was collected before they underwent testicular sperm extraction (TESE) operation. The metabolomic fingerprinting was evaluated by Raman spectrometer. Principal component analysis (PCA) and an unsupervised statistical method, was used to detect outliers and find the structure of the data. The PCA was analyzed by MATLAB software. RESULTS: Metabolic fingerprinting of seminal plasma from NOA showed that TESE (+) versus TESE(-) patients were classified by PCA. Furthermore, a possible subdivision of TESE(-) group was observed. Additionally, TESE(-) patients were in extreme oxidative imbalance compared to TESE(+) patients. CONCLUSION: Metabolic fingerprinting of seminal plasma can be considered as a breakthrough, an easy and cheap method for prediction presence of spermatogenesis in NOA.

Inhibitory action of dicyclomine on lipase activity, kinetics and molecular study.

Lipase is one of the most important groups of enzymes for industry and medicine. It breaks down triacylglycerol to glycerol and fatty acids. Some bacteria use lipase to degrade the extracellular matrix of the host cells to penetrate into the tissues. Dicyclomine is a muscarinic antagonist receptor that relieves the smooth muscle spasm of the gastrointestinal tract and affects the cardiovascular system. In this research, the effect of a dicyclomine on the lipase activity of Pseudomonas aeruginosa was studied. Hanes-Woolf plot showed that the drug inhibited the enzyme by competitive inhibition. The IC50 value (60uM) and Ki (30uM) of the drug revealed that the drug bound to enzyme with high affinity. Determination of enzyme activity in various temperature showed that the maximum activity of lipase was at 60°C both in the presence and absence of the drug. Arrhenius plot determined that the activation energy of the enzyme reaction was increased in the presence of the drug. The model of binding demonstrated that the drug entered a pocket containing 10 amino acids and interacted by hydrogen bond and hydrophobic interaction and the conformational change of the enzyme after binding of the drug was confirmed by fluorescence measurement.

The Challenge of Human Spermatozoa Proteome: A Systematic Review.

Currently, there are 20,197 human protein-coding genes in the most expertly curated database (UniProtKB/Swiss-Pro). Big efforts have been made by the international consortium, the Chromosome-Centric Human Proteome Project (C-HPP) and independent researchers, to map human proteome. In brief, anno 2017 the human proteome was outlined. The male factor contributes to 50% of infertility in couples. However, there are limited human spermatozoa proteomic studies. Firstly, the development of the mapping of the human spermatozoa was analyzed. The human spermatozoa have been used as a model for missing proteins. It has been shown that human spermatozoa are excellent sources for finding missing proteins. Y chromosome proteome mapping is led by Iran. However, it seems that it is extremely challenging to map the human spermatozoa Y chromosome proteins based on current mass spectrometry-based proteomics technology. Post-translation modifications (PTMs) of human spermatozoa proteome are the most unexplored area and currently the exact role of PTMs in male infertility is unknown. Additionally, the clinical human spermatozoa proteomic analysis, anno 2017 was done in this study.

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.

Untargeted metabolomic profiling of seminal plasma in nonobstructive azoospermia men: A noninvasive detection of spermatogenesis.

Male factor infertility is involved in almost half of all infertile couples. Lack of the ejaculated sperm owing to testicular malfunction has been reported in 6-10% of infertile men, a condition named nonobstructive azoospermia (NOA). In this study, we investigated untargeted metabolomic profiling of the seminal plasma in NOA men using gas chromatography-mass spectrometry and advance chemometrics. In this regard, the seminal plasma fluids of 11 NOA men with TESE-negative, nine NOA men with TESE-positive and 10 fertile healthy men (as a control group) were collected. Quadratic discriminate analysis (QDA) technique was implemented on total ion chromatograms (TICs) for identification of discriminatory retention times. We developed multivariate classification models using the QDA technique. Our results revealed that the developed QDA models could predict the classes of samples using their TIC data. The receiver operating characteristic curves for these models were >0.88. After recognition of discriminatory retention time's asymmetric penalized least square, evolving factor analysis, correlation optimized warping and alternating least squares strategies were applied for preprocessing and deconvolution of the overlapped chromatographic peaks. We could identify 36 discriminatory metabolites. These metabolites may be considered discriminatory biomarkers for different groups in NOA.

Inhibition of pseudoperoxiadse activity of human red blood cell hemoglobin by methocarbamol.

After red blood cells lysis, hemoglobin is released to blood circulation. Hemoglobin is carried in blood by binding to haptoglobin. In normal individuals, no free hemoglobin is observed in the blood, because most of the hemoglobin is in the form of haptoglobin complex. In some diseases that are accompanied by hemolysis, the amount of released hemoglobin is higher than its complementary haptoglobin. As a result, free hemoglobin appears in the blood, which is a toxic compound for these patients and may cause renal failure, hypertensive response and risk of atherogenesis. Free hemoglobin has been determined to have peroxidase activity and considered a pseudoenzyme. In this study, the effect of methocarbamol on the peroxidase activity of human hemoglobin was investigated. Our results showed that the drug inhibited the pseudoenzyme by un-competitive inhibition. Both Km and Vmax decreased by increasing the drug concentration. Ki and IC50 values were determined as 6 and 10mM, respectively. Docking results demonstrated that methocarbamol did not attach to heme group directly. A hydrogen bond linked NH2 of carbamate group of methocarbamol to the carboxyl group of Asp126 side chain. Two other hydrogen bonds could be also observed between hydroxyl group of the drug and Ser102 and Ser133 residues of the pseudoenzyme.

Binding of Cimetidine to Balb/C Mouse Liver Catalase; Kinetics and Conformational Studies.

Catalase is responsible for converting hydrogen peroxide (H2O2) into water and oxygen in cells. This enzyme has high affinity for hydrogen peroxide and can protect the cells from oxidative stress damage. Catalase is a tetramer protein and each monomer contains a heme group. Cimetidine is a histamine H2 receptor blocker which inhibits acid release from stomach and is used for gasterointestinal diseases. In this research, effect of cimetidine on the activity of liver catalase was studied and the kinetic parameters of this enzyme and its conformational changes were investigated. Cell free extract of mouse liver was used for the catalase assay. The activity of the catalase was detected in the absence and presence of cimetidine by monitoring hydrogen peroxide reduction absorbance at 240 nm. The purified enzyme was used for conformational studies by Fluorescence spectrophotometry. The data showed that cimetidine could inhibit the enzyme in a non-competitive manner. Ki and IC50 values of the drug were determined to be about 0.75 and 0.85 uM, respectively. The Arrhenius plot showed that activation energy was 6.68 and 4.77 kJ/mol in the presence and absence of the drug, respectively. Fluorescence spectrophotometry revealed that the binding of cimetidine to the purified enzyme induced hyperchromicity and red shift which determined the conformational change on the enzyme. Cimetidine could non-competitively inhibit the liver catalase with high affinity. Binding of cimetidine to the enzyme induced conformational alteration in the enzyme.

Exploring the human seminal plasma proteome: an unexplored gold mine of biomarker for male infertility and male reproduction disorder.

BACKGROUND: The human seminal fluid is a complex body fluid. It is not known how many proteins are expressed in the seminal plasma; however in analog with the blood it is possible up to 10,000 proteins are expressed in the seminal plasma. The human seminal fluid is a rich source of potential biomarkers for male infertility and reproduction disorder. METHODS: In this review, the ongoing list of proteins identified from the human seminal fluid was collected. To date, 4188 redundant proteins of the seminal fluid are identified using different proteomics technology, including 2-DE, SDS-PAGE-LC-MS/MS, MudPIT. However, this was reduced to a database of 2168 non-redundant protein using UniProtKB/Swiss-Prot reviewed database. RESULTS: The core concept of proteome were analyzed including pI, MW, Amino Acids, Chromosome and PTM distribution in the human seminal plasma proteome. Additionally, the biological process, molecular function and KEGG pathway were investigated using DAVID software. Finally, the biomarker identified in different male reproductive system disorder was investigated using proteomics platforms so far. CONCLUSION: In this study, an attempt was made to update the human seminal plasma proteome database. Our finding showed that human seminal plasma studies used to date seem to have converged on a set of proteins that are repeatedly identified in many studies and that represent only a small fraction of the entire human seminal plasma proteome.

Correction: Knockdown of the Sodium-Dependent Phosphate Co-Transporter 2b (NPT2b) Suppresses Lung Tumorigenesis.

[This corrects the article DOI: 10.1371/journal.pone.0077121.].

Gene Expression and Pulmonary Toxicity of Chitosan-graft- Polyethylenimine as Aerosol Gene Carrier.

Chitosan-graft-polyethylenimine (CHI-g-PEI) copolymer has been used for the improvement of low transfection efficiency of chitosan. The present study aims to test the pulmonary toxicity and efficiency of CHI-g-PEI as an aerosol gene carrier. Mice were exposed to aerosol containing green-fluorescent protein (GFP)-polyethylenimine (PEI) or GFP-CHI-g-PEI complexes for 30 min during the development of our nose-only exposure chamber (NOEC) system. CHI-g-PEI-mediated aerosol delivery demonstrated 15.65% enhancement of the fluorescence intensity. Compared to PEI, CHI-g-PEI showed no significant pulmonary toxicity. In summary, using CHI-g-PEI is safe and shows high transfection in aerosol gene delivery to animals, and enhanced efficiency was achieved through our aerosol gene delivery system. Therefore, CHI-g-PEI and this system would be applicable to future study for aerosol gene therapy.