Treatment with Radiopharmaceuticals and Radionuclides in Breast Cancer: Current Options

Radiopharmaceutical therapy (RPT) is an effective and safe treatment for many types of cancer. RPT acts by binding radioactive atoms to tumor-associated antigens, monoclonal antibodies, nanoparticles, peptides, and small molecules. These treatments ensure that a concentrated dose is delivered to the targeted tumor tissue while preserving the normal tissues surrounding the tumor. Given these features, RPT is superior to traditional methods. This review article aimed to performa comprehensive review and evaluation of the potential of radionuclides and radiopharmaceuticals used in breast cancer treatment in preclinical studies conducted in the last five years

Radiopharmaceuticals developed for Zr-89-Immuno-PET

The development of molecular imaging agents used for imaging of cancer tissue is of great importance for the early detection of cancer. Positron emission tomography (PET) radiopharmaceuticals consists of a positron-emitting radionuclide and a molecularstructure. Zr-89-labeled monoclonal antibodies (mAbs), peptides, nanoparticles, proteins, and other compounds are called Zr-89-Immuno-PET and are used in cancer tissue imaging. This review provides a general overview of the potential of molecules labeled with Zr-89 radionuclide, which is chosen due to its long half-life, in preclinical and clinical studies. In light of these studies, radiopharmaceuticals created using nanoparticles have greater potential than those using antibodies

In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus.

A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F). Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-β-N-acetylglucosaminidase (EC3.2.1.96, Endo H), another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes

Engineering, and production of functionally active human Furin in N. benthamiana plant: In vivo post-translational processing of target proteins by Furin in plants.

A plant expression platform with eukaryotic post-translational modification (PTM) machinery has many advantages compared to other protein expression systems. This promising technology is useful for the production of a variety of recombinant proteins including, therapeutic proteins, vaccine antigens, native additives, and industrial enzymes. However, plants lack some of the important PTMs, including furin processing, which limits this system for the production of certain mammalian complex proteins of therapeutic value. Furin is a ubiquitous proprotein convertase that is involved in the processing (activation) of a wide variety of precursor proteins, including blood coagulation factors, cell surface receptors, hormones and growth factors, viral envelope glycoproteins, etc. and plays a critical regulatory role in a wide variety of cellular events. In this study, we engineered the human furin gene for expression in plants and demonstrated the production of a functional active recombinant truncated human furin in N. benthamiana plant. We demonstrate that plant produced human furin is highly active both in vivo and in vitro and specifically cleaved the tested target proteins, Factor IX (FIX) and Protective Antigen (PA83). We also demonstrate that both, enzymatic deglycosylation and proteolytic processing of target proteins can be achieved in vivo by co-expression of deglycosylating and furin cleavage enzymes in a single cell to produce deglycosylated and furin processed target proteins. It is highly expected that this strategy will have many potential applications in pharmaceutical industry and can be used to produce safe and affordable therapeutic proteins, antibodies, and vaccines using a plant expression system

Evaluation of the deglycosylation efficiency of Pfs48/45-10C by Endo H or PNGase F <i>in vivo</i>.

<p>Pfs48/45-10C (A), Pfs48/45 (B) and PA83 (C) were co-expressed with Endo H or PNGase F at different ratios of OD600 of Agrobacteria carrying Endo H, PNGase F and target genes, as indicated. The efficiency of deglycosylation of target proteins by Endo H or PNGase F was evaluated by Western blot analysis. Size reduction of Pfs48/45-10C, Pfs48/45 and PA83 show as an indicator of glycan removal. Proteins were probed with the anti-4xHis Tag mAb (BioLegend) (A,C) or anti-FLAG antibody (B). Indications of proteins in figures are the same as shown above.</p

Western blot analysis of bacterial Endo H or PNGase F produced in <i>Nicotiana benthamiana</i> plants.

<p><i>N</i>. <i>benthamiana</i> plants were infiltrated with pBI-Endo H or pBI-PNGase F constructs to produce Endo H or PNGase F. Lanes: 1-crude extract prepared from control plant; 2- crude extract prepared from plant infiltrated with bacterial Endo H (pBI-Endo H) and 10, 20 and 40 fold diluted samples were loaded into gel; 3- crude extract prepared from plant infiltrated with bacterial PNGase F (pBI-PNGase F),and 2, 5 or 10 fold diluted samples were loaded into gel; 4- purified plant produced Endo H used as a standard protein; 10 or 25 ng were loaded into gel. M: MagicMark XP Western Protein Standard (ThermoFisher Scientific).</p

Western blot analysis of co-expression <i>Bacillus anthracis</i> PA83 (A), Pfs48/45 (B) and Pfs48/45-10C with bacterial Endo H or PNGase F in <i>N</i>. <i>benthamiana</i> plants.

<p>(A) Western blot analysis of co-expression of PA83. Lanes: 1- <i>N</i>. <i>benthamiana</i> plant was infiltrated with pBI-PA83 construct, for the production of glycosylated PA83, 2,3- <i>N</i>. <i>benthamiana</i> plants were infiltrated with combinations of the pBI-Endo H/pBI-PA83 or pBI-PNGase F/pBI-PA83 constructs, for the production of Endo H (2) or PNGase F (3) deglycosylated PA83 proteins. (B) Western blot analysis of co-expression of Pfs48/45. Lanes: 1-<i>N</i>. <i>benthamiana</i> plant was infiltrated with pEAQ-Pfs48/45 construct for the production of glycosylated Pfs48/45;2,3- <i>N</i>. <i>benthamiana</i> plants were infiltrated with combinations of the pBI-Endo H/pEAQ-Pfs48/45 or pBI-PNGase F/pEAQ-Pfs48/45constructs for the production of Endo H (2) and PNGase F (3) deglycosylated Pfs48/45 proteins. (C) Western blot analysis of co-expression of Pfs48/45-10C. Lanes: 1- <i>N</i>. <i>benthamiana</i> plant was infiltrated with pEAQ-Pfs48/45-10C construct for the production of glycosylated Pfs48/45-10C; 2,3- <i>N</i>. <i>benthamiana</i> plants were infiltrated with combinations of the pBI-Endo H/pEAQ-Pfs48/45 or pBI-PNGase F/pEAQ-Pfs48/45constructs for the production of Endo H (2) and PNGase F (3) deglycosylated Pfs48/45-10C proteins. gPA83- glycosylated PA83; dPA83- deglycosylated PA83; gPfs48/45: glycosylated Pfs48/45; dPfs48/45: deglycosylated Pfs48/45; gPfs48/45-10C: glycosylated Pfs48/45-10C; dPfs48/45-10C: deglycosylated Pfs48/45-10C.M: MagicMark XP Western Protein Standard (ThermoFisher Scientific). PA83 proteins were detected using the anti-Bacillus anthracis protective antigen antibody BAP0101 (Cat. No. ab1988, Abcam); Ps48/45, Endo H or PNGase F proteins were detected using the anti-FLAG antibody (BioLegend). Pfs48/45-10C protein was detected using the purified anti-His Tag antibody (Cat. No. 652502, BioLegend).</p

Study of stability of glycosylated and deglycosylated PA83 variants using SDS-PAGE analysis.

<p>Plant produced, glycosylated PA83, and <i>in vivo</i> Endo H or PNGase F deglycosylated forms of PA83 were purified as described in Materials and Methods. (A) The purified plant produced PA83 variants were stored for 1 hour at 37°C or for 72 hours at 4°C and analyzed by SDS-PAGE. Lanes were loaded with ~8.0 μg per lane for glycosylated, Endo H or PNGase F <i>in vivo</i> deglycosylated plant produced PA83 proteins. (B) Purified, plant produced, glycosylated PA83, and <i>in vivo</i> deglycosylated (co-expressed with Endo H or PNGase F) and <i>in vitro</i> deglycosylated (by commercial Endo H) proteins were incubated at 37°C for 1, 4, 8, 16 and 24 hours, and analyzed in SDS-PAGE. Lanes were loaded with ~5.0 μg per lane for each sample. M- color prestained protein standard (New England Biolabs).</p

Schematic representation of Endo H or PNGase F cleavages.

<p>(A) Endo H cleaves between the two GlcNAc residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one GlcNAc remaining on the asparagines (Asn). (B) Peptide -<i>N</i>-Glycosidase F (PNGase F), is an amidase that cleaves the bond between GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from <i>N</i>-linked glycoproteins;</p

Forensic child and adolescent psychiatry: From field experiences to education standards

Objective: Forensic evaluation of children is one of the most problematic areas of child and adolescent psychiatry. In this study we aimed to examine Turkish Child and adolescent psychiatrists' attitudes and problems in forensic psychiatry