scholarly journals Development of affinity beads-based in vitro metal-ligand binding assay reveals dominant cadmium affinity of thiol-rich small peptides phytochelatins beyond glutathione

2021 ◽  
Author(s):  
Shimpei Uraguchi ◽  
Kenichiro Nagai ◽  
Fumii Naruse ◽  
Yuto Otsuka ◽  
Yuka Ohshiro ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Binding Assay ◽  
Emission Spectrometry ◽  
Dependent Manner ◽  
Icp Oes ◽  
Small Peptides ◽  
Competition Assay ◽  
Metal Ligand ◽  
Affinity Beads

AbstractFor a better understanding of metal-ligand interaction and its function in cells, we developed an easy, sensitive, and high-throughput method to quantify ligand-metal(loid) binding affinity under physiological conditions, by combining ligand-attached affinity beads and inductively coupled plasma-optical emission spectrometry (ICP-OES). Glutathione (GSH) and two phytochelatins (PC2 and PC3, small peptides with different thiol numbers) were employed as model ligands and attached to affinity beads. The principal of the assay resembles that of affinity purification of proteins in biochemistry: metals binding to the ligand on the beads and the rest in the buffer are separated by a spin-column and quantified by ICP-OES. The binding assay using the GSH-attached beads and various metal(loid)s suggested that the assay can detect the different affinity of the metal-ligand interactions, in accordance with the order of the Irving–Williams series and the reported stability constants. The binding assay using PC2 or PC3-attached beads suggested positive binding between PCs and nickel, copper, zinc, cadmium, and arsenite ions in a thiol-dependent manner. We then conducted the competition assay using cadmium, manganese, iron, copper, and zinc and the results suggested a better binding affinity of PC2 with cadmium than with the essential metals. Another competition assay using PC2 and GSH suggested a robust binding affinity between PCs and cadmium compared to GSH and cadmium. These results suggested the dominance of PC-Cd complex formation in vitro, supporting the physiological importance of PCs for the detoxification of cadmium in vivo. We also discuss the further potential application of the assay.

scholarly journals Octreotide-conjugated silver nanoparticles for active targeting of somatostatin receptors and their application in a nebulized rat model

2021 ◽  
Vol 11 (1) ◽  
pp. 266-283
Author(s):  
Ahmed A. H. Abdellatif ◽  
Riaz A. Khan ◽  
Ahmad H. Alhowail ◽  
Abdulmajeed Alqasoumi ◽  
Sultan M. Sajid ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Silver Nanoparticles ◽  
Cell Lines ◽  
Somatostatin Receptors ◽  
Somatostatin Analogs ◽  
Drug Uptake ◽  
Emission Spectrometry ◽  
Dependent Manner

Abstract Drug uptake and distribution through cell–receptor interactions are of prime interest in reducing the adverse effects and increasing the therapeutic effectiveness of delivered formulations. This study aimed to formulate silver nanoparticles (AgNPs) conjugated to somatostatin analogs for specific delivery through somatostatin receptors (SSTRs) expressed on cells and by nebulizing the prepared AgNPs formulations into lung cells for in vivo application. AgNPs were prepared using the citrate reduction method, yielding AgNPs–CTT, which was further chemically conjugated to octreotide (OCT) to form AgNPs–OCT through an amide linkage. The AgNPs–OCT formulation was coated using alginate to yield a carrier, AgNPs–OCT–Alg, feasible for drug delivery through nebulization. AgNPs were uniform in size with an acceptable range of zeta potential. Furthermore, the concentrations of AgNP formulations were found safe for the model cell lines used, and cell proliferation was significantly reduced in a dose-dependent manner (p < 0.05). In the healthy lung tissues, AgNPs–OCT–Alg accumulated at a concentration of 0.416 ± 5.7 mg/kgtissue, as determined via inductively coupled plasma optical emission spectrometry. This study established the accumulation of AgNPs, specifically the AgNPs–OCT–Alg, in lung tissues, and substantiated the active, specific, and selective targeting of SSTRs at pulmonary sites. The anticancer efficacy of the formulations was in vitro tested and confirmed in the MCF-7 cell lines. Owing to the delivery suitability and cytotoxic effects of the AgNPs–OCT–Alg formulation, it is a potential drug delivery formulation for lung cancer therapy in the future.

scholarly journals An optimal anti-tumor response by a novel CEA/CD3 bispecific antibody for colorectal cancers

2021 ◽  
Author(s):  
Ninghai Wang ◽  
Harshal Patel ◽  
Irene Schneider ◽  
Xin Kai ◽  
Avanish K Varshney ◽  
...  
Keyword(s):  
T Cell ◽  
Binding Affinity ◽  
Tumor Antigen ◽  
Xenograft Model ◽  
Variable Region ◽  
Dependent Manner ◽  
Cytokine Release ◽  
Complex Development

Abstract Background CD3-based bispecific T cell engagers (bsTCEs) are one of the most promising bispecific antibodies for effective cancer treatments. To elicit target-specific T cell-mediated cytotoxicity, these bsTCEs contain at least one binding unit directed against a tumor antigen and another binding unit targeting CD3 in T cell antigen receptor complex. Development of CD3-based bsTCEs, however, has been severely hampered by dose limiting toxicities due to cytokine release syndrome. To address this limitation, we developed a novel functionally trivalent TCE (t-TCE) antibody containing affinity reduced CD3 binding unit, positioned to ensure monovalent CD3 engagement, in combination with bivalent tumor antigen binding of Carcinoembryonic Antigen (CEA). Methods We modeled the variable region of anti-CD3 in the CDRs of the heavy chain and obtained CD3 binders with reduced binding affinity. Two optimized versions CEA/CD3-v1 and CEA/CD3-v2 were identified and generated in tetravalent format, characterized and compared in vitro and in vivo. Results Our lead candidate, CEA/CD3-v2, demonstrated sub-nanomolar binding and picomolar potency against a panel of CEA-expressing cancer cell lines. In addition, we detected reduced T cell cytokine release with potent cytotoxic activity. Our t-TCE CEA/CD3-v2 molecule demonstrated strong anti-tumor effect in a dose dependent manner in human PBMC xenograft model. Furthermore, combination of CEA/CD3-v2 with atezolizumab provided synergistic antitumor effect. Conclusions Because of effective tumor cell killing with various level of CEA expression and reduced cytokine release, CEA/CD3 BsTCE may greatly benefit in CEA positive cancer immunotherapy. Statement of Significance. Through optimization of CD3 binding affinity and tetravalent format with functional monovalent binding to CD3, t-TCE CEA/CD3–2 molecule not only retains high potency in vitro and in vivo, but also significantly reduces cytokine release.

scholarly journals The Arabidopsis MIK2 receptor elicits immunity by sensing a conserved signature from phytocytokines and microbes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shuguo Hou ◽  
Derui Liu ◽  
Shijia Huang ◽  
Dexian Luo ◽  
Zunyong Liu ◽  
...  
Keyword(s):  
Immune Responses ◽  
Dependent Manner ◽  
Receptor Kinase ◽  
Small Peptides ◽  
Robust Immune Response ◽  
Repeat Domain ◽  
Signature Peptides ◽  
Cytosolic Receptor

AbstractSessile plants encode a large number of small peptides and cell surface-resident receptor kinases, most of which have unknown functions. Here, we report that the Arabidopsis receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) recognizes the conserved signature motif of SERINE-RICH ENDOGENOUS PEPTIDEs (SCOOPs) from Brassicaceae plants as well as proteins present in fungal Fusarium spp. and bacterial Comamonadaceae, and elicits various immune responses. SCOOP signature peptides trigger immune responses and altered root development in a MIK2-dependent manner with a sub-nanomolar sensitivity. SCOOP12 directly binds to the extracellular leucine-rich repeat domain of MIK2 in vivo and in vitro, indicating that MIK2 is the receptor of SCOOP peptides. Perception of SCOOP peptides induces the association of MIK2 and the coreceptors SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (SERK3) and SERK4 and relays the signaling through the cytosolic receptor-like kinases BOTRYTIS-INDUCED KINASE 1 (BIK1) and AVRPPHB SUSCEPTIBLE1 (PBS1)-LIKE 1 (PBL1). Our study identifies a plant receptor that bears a dual role in sensing the conserved peptide motif from phytocytokines and microbial proteins via a convergent signaling relay to ensure a robust immune response.

scholarly journals Phosphorylation of Connexin36 near the C-terminus switches binding affinities for PDZ-domain and 14–3–3 proteins in vitro

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Stephan Tetenborg ◽  
Helen Y. Wang ◽  
Lena Nemitz ◽  
Anne Depping ◽  
Alexsandra B. Espejo ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Binding Affinity ◽  
Protein Microarray ◽  
Pdz Domain ◽  
Phosphorylation Site ◽  
Binding Domain ◽  
Dependent Manner ◽  
C Terminus ◽  
Activity Dependent

Abstract Connexin36 (Cx36) is the most abundant connexin in central nervous system neurons. It forms gap junction channels that act as electrical synapses. Similar to chemical synapses, Cx36-containing gap junctions undergo activity-dependent plasticity and complex regulation. Cx36 gap junctions represent multimolecular complexes and contain cytoskeletal, regulatory and scaffolding proteins, which regulate channel conductance, assembly and turnover. The amino acid sequence of mammalian Cx36 harbors a phosphorylation site for the Ca2+/calmodulin-dependent kinase II at serine 315. This regulatory site is homologous to the serine 298 in perch Cx35 and in close vicinity to a PDZ binding domain at the very C-terminal end of the protein. We hypothesized that this phosphorylation site may serve as a molecular switch, influencing the affinity of the PDZ binding domain for its binding partners. Protein microarray and pulldown experiments revealed that this is indeed the case: phosphorylation of serine 298 decreased the binding affinity for MUPP1, a known scaffolding partner of connexin36, and increased the binding affinity for two different 14–3–3 proteins. Although we did not find the same effect in cell culture experiments, our data suggest that phosphorylation of serine 315/298 may serve to recruit different proteins to connexin36/35-containing gap junctions in an activity-dependent manner.

scholarly journals The effect of different doses of radiation on the morphogical, mechanical and chemical properties of the primary and permanent teeth—an in vitro study

2020 ◽  
Author(s):  
Gülsüm Duruk ◽  
Burçin Acar ◽  
Öztun Temelli
Keyword(s):  
Chemical Properties ◽  
Neck Region ◽  
Permanent Teeth ◽  
Emission Spectrometry ◽  
Icp Oes ◽  
Sem Images ◽  
Primary Molar ◽  
Human Teeth ◽  
And Chemical Properties

Abstract Background. Radiotherapy, applied to the head and neck region, can cause radiation side effects such as reduction of saliva and radiation caries. The aim of this study was to perform an in vitro assessment of the effects of radiation therapy on the morphological, mechanical, and chemical properties of primary and permanent teeth. Methods. 92 extracted human teeth (46 impacted wisdom teeth, 46- primary molar teeth) were used. The teeth were divided into two directions: the mesiodistal direction and the vestibulolingual direction. The vestibular sections were used for vickers analysis, and lingual sections were used for Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) analysis. The teeth in the experimental group were fixed to wax models. Each model had an equal number of teeth (n=7). The doses were applied to the teeth for 6 weeks; 5 week days and 2Gy daily. After the radiotherapy conducted weekly, a wax model was taken from radiation reception. Along with elemental contents (Ca, P, Na, Mg, P) of the teeth, enamel and dentin microhardness were evaluated, and SEM analyzes were performed on 8 teeth. These teeth were compared with the other non-irradiated sections. Results. Radiation caused a decrease in microhardness of enamel and dentin(p<0.05). In elemental analysis by ICP-OES, it was observed that there were decreases in all doses, while the highest decrease was in 60Gy dose(p<0.01). In the experimental groups, amorphous structures were encountered in SEM images. Conclusions. Radiation has negative effects on the teeth structure and additional studies are needed in this regard. This study indicates that radiotherapy patients are at a higher risk for dental caries

scholarly journals The effect of different doses of radiation on the morphogical, mechanical and chemical properties of the primary and permanent teeth—an in vitro study

2020 ◽  
Author(s):  
Gülsüm Duruk ◽  
Burçin Acar ◽  
Öztun Temelli
Keyword(s):  
Chemical Properties ◽  
Neck Region ◽  
Permanent Teeth ◽  
Emission Spectrometry ◽  
Icp Oes ◽  
Sem Images ◽  
Primary Molar ◽  
Human Teeth ◽  
And Chemical Properties

Abstract Background. Radiotherapy, applied to the head and neck region, can cause radiation side effects such as reduction of saliva and radiation caries. The aim of this study was to perform an in vitro assessment of the effects of radiation therapy on the morphological, mechanical, and chemical properties of primary and permanent teeth. Methods. 92 extracted human teeth (46 impacted wisdom teeth, 46- primary molar teeth) were used. The teeth were divided into two directions: the mesiodistal direction and the vestibulolingual direction. The vestibular sections were used for vickers analysis, and lingual sections were used for Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) analysis. The teeth in the experimental group were fixed to wax models. Each model had an equal number of teeth (n=7). The doses were applied to the teeth for 6 weeks; 5 week days and 2Gy daily. After the radiotherapy conducted weekly, a wax model was taken from radiation reception. Along with elemental contents (Ca, P, Na, Mg, P) of the teeth, enamel and dentin microhardness were evaluated, and SEM analyzes were performed on 8 teeth. These teeth were compared with the other non-irradiated sections. Results. Radiation caused a decrease in microhardness of enamel and dentin(p<0.05). In elemental analysis by ICP-OES, it was observed that there were decreases in all doses, while the highest decrease was in 60Gy dose(p<0.01). In the experimental groups, amorphous structures were encountered in SEM images. Conclusions. Radiation has negative effects on the teeth structure and additional studies are needed in this regard. This study indicates that radiotherapy patients are at a higher risk for dental caries

scholarly journals The effect of different doses of radiation on the morphogical, mechanical and chemical properties of the primary and permanent teeth—an in vitro study

2020 ◽  
Author(s):  
Gülsüm Duruk ◽  
Burçin Acar ◽  
Öztun Temelli
Keyword(s):  
Chemical Properties ◽  
Neck Region ◽  
Permanent Teeth ◽  
Emission Spectrometry ◽  
Icp Oes ◽  
Sem Images ◽  
Primary Molar ◽  
Human Teeth ◽  
And Chemical Properties

Abstract Background. Radiotherapy, applied to the head and neck region, can cause radiation side effects such as reduction of saliva and radiation caries. The aim of this study was to perform an in vitro assessment of the effects of radiation therapy on the morphological, mechanical, and chemical properties of primary and permanent teeth.Methods. 92 extracted human teeth (46 impacted wisdom teeth, 46 primary molar teeth) were used. The teeth were divided into two directions: the mesiodistal direction and the vestibulolingual direction. The vestibular sections were used for the vickers analysis, and lingual sections were used for the Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) analysis. The teeth in the experimental group were fixed to wax models. Each model had an equal number of teeth (n=7). The doses were applied to the teeth for 6 weeks; 5 week days and 2Gy daily. After the radiotherapy was conducted weekly, a wax model was taken from radiation reception. Along with the elemental contents (Na, K, Mg, P, and Ca) of the teeth, enamel and dentin microhardness was evaluated, and SEM analyzes were performed on 8 teeth. These teeth were compared with the other non-irradiated sections. Results. Radiation caused a decrease in microhardness of enamel and dentin(p<0.05). In the elemental analysis by ICP-OES, it was observed that there were decreases in all doses, while the highest decrease was in 60Gy dose(p<0.01). In the experimental groups, amorphous structures were encountered in SEM images.Conclusions. Radiation has negative effects on the teeth structure and additional studies are needed in this regard. This study indicates that radiotherapy patients are at a higher risk for dental caries.

scholarly journals Simplified ICP OES-Based Method for Determination of 12 Elements in Commercial Bottled Birch Saps: Validation and Bioaccessibility Study

Molecules ◽  
2020 ◽  
Vol 25 (5) ◽  
pp. 1256
Author(s):  
Maja Welna ◽  
Anna Szymczycha-Madeja ◽  
Pawel Pohl
Keyword(s):  
Inductively Coupled Plasma ◽  
Optical Emission ◽  
Direct Analysis ◽  
Emission Spectrometry ◽  
Dietary Intakes ◽  
Icp Oes ◽  
Element Analysis ◽  
Inductively Coupled

Commercially bottled birch saps (BSs) were analyzed for several nutrient (Ca, Cu, Fe, Mg, and Zn) and toxic (As, Cd, Ni, and Pb) elements using inductively coupled plasma optical emission spectrometry (ICP OES). The method was validated under the conditions of several sample preparation procedures, including a traditional digestion as well as alternative non-digestion schemes. It was found that the direct analysis of untreated BSs gives the best results, i.e., limits of detection at 0.02–5.8 ng mL−1, precision better than 5%, accuracy from 98.0% to 104.5% and determination of 12 elements in a short time (~1 min per sample). The multi-element analysis of nine commercially available bottled BSs showed that they contained mainly Mg and Ca, small quantities of Mn, Zn, Cu, and Fe, but are free from toxic elements such as As, Cd, Ni, and Pb. Additionally, the nutritional value of BSs was examined using in vitro gastro-intestinal digestion (GID) to determine the bioaccessible fraction of elements. Accordingly, bioaccessibility of nutritious ones (Ca, Cu, Fe, Mg, Zn) was <40%. Drinking daily 1 L of BSs covered <2.5% of recommended dietary intakes (RDIs) of the aforementioned elements. Only the bioaccessibility of Mn highly contributes to its RDI.

Identification of promoter PcadR, in silico characterization of cadmium resistant gene cadR and molecular cloning of promoter PcadR from Pseudomonas aeruginosa BC15

2018 ◽  
Vol 34 (12) ◽  
pp. 819-833 ◽  
Author(s):  
Rajkumar Prabhakaran ◽  
Sebastin Nirmal Rajkumar ◽  
Tharmarajan Ramprasath ◽  
Govindan Sadasivam Selvam
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Cloning ◽  
Binding Affinity ◽  
In Silico ◽  
Dependent Manner ◽  
Atomic Contact ◽  
In Silico Studies ◽  
Metal Sensing

Cadmium (Cd) remediation in Pseudomonas aeruginosa is achieved through the function of two vital genes, cadA and cadR, that code for P-type ATPase (CadA) and transcription regulatory protein (CadR), respectively. Although numerous studies are available on these metal-sensing and regulatory proteins, the promoter of these genes, metal sensing and binding ability, are poorly understood. The present work is aimed at the characterization of the CadR protein, identification of the P cadR promoter and protein–promoter–metal binding affinity using bioinformatics and to validate the results by cloning the P cadR promoter in Escherichia coli DH5α. The promoter regions and its curvature were identified and analysed using PePPER software (University of Groningen, The Netherland) and the Bendit program (Version: v.1.0), respectively. Using Phyre, the three-dimensional structure of CadR was modelled, and the structure was validated by Ramachandran plots. The DNA-binding domain was present in the N-terminal region of CadR. A dimeric interface was observed in helix-turn-helix and metal ion-binding sites at the C-terminal. Docking studies showed higher affinity of Cd to both CadR (Atomic contact energy = −15.04 kcal/Mol) and P cadR (Atomic contact energy = −40.18 kcal/Mol) when compared to other metal ions. CadR with P cadR showed the highest binding affinity (Atomic contact energy= −250.40 kcal/Mol) when compared with P cadA. In vitro studies using green fluorescent protein tagged with P cadR ( gfp-P cadR) cloned in E. coli-expressed gfp protein in a concentration-dependent manner upon Cd exposure. Based on our in silico studies and in vitro molecular cloning analysis, we conclude that P cadR and CadR are active only in the presence of Cd. The CadR protein has the highest binding affinity with P cadR. As it became apparent that the cadR gene regulates the P cadR activity in the presence of Cd with high specificity, and the cadR and P cadR can be used as a biological tool for development of a microbial biosensor.

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