Cadmium SAD phasing at CuKα wavelength [version 1; peer review: 2 approved]

Single-wavelength anomalous diffraction (SAD) is the most common method for de novo elucidation of macromolecular structures by X-ray crystallography. It requires an anomalous scatterer in a crystal to calculate phases. A recent study by Panneerselvam et al. emphasized the utility of cadmium ions for SAD phasing at the standard synchrotron wavelength of 1 Å. Here we show that cadmium is also useful for phasing of crystals collected in-house with CuKα radiation. Using a crystal of single-domain antibody as an experimental model, we demonstrate how cadmium SAD can be conveniently employed to solve a CuKα dataset. We then discuss the factors which make this method generally applicable

Image_1_Isolation of living dopaminergic neurons labeled with a fluorescent ligand of the dopamine transporter from mouse substantia nigra as a new tool for basic and applied research.JPEG

Dopaminergic neurons (DNs) of the nigrostriatal system control the motor function, and their degeneration leads to the development of Parkinson’s disease (PD). A stumbling block in the study of DNs in the whole substantia nigra (SN) is the lack of tools to analyze the expression of most of the genes involved in neurotransmission, neurodegeneration, and neuroplasticity, since they are also expressed in other cells of the SN. Therefore, this study aimed to develop a fluorescence-activated cell sorting method for isolating living DNs from the SN of wild-type mice using two fluorescent dyes, DRAQ5 (nuclear stain) and a dopamine uptake inhibitor GBR 12909 coupled to a fluorophore (DN stain). We have developed a method for selecting a population of DNs from the SN of mice, as evidenced by: (i) immunopositivity of 95% of the sorted cells for tyrosine hydroxylase, the first enzyme of dopamine synthesis; (ii) the sorted cells expressing the genes for specific proteins of the dopaminergic phenotype, tyrosine hydroxylase, the dopamine transporter, and vesicular monoamine transporter 2 and non-specific proteins, such as aromatic L-amino acid decarboxylase, non-specific enzyme of dopamine synthesis. We then compared the changes in gene expression found in the sorted DNs and in the SN homogenate in a PD model we developed, reproduced in mice by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Using quantitative PCR, we obtained evidence of the same changes in the expression of specific genes in the sorted DNs of SN and in the SN homogenate of a MPTP mouse model of PD, compared with the control. The undoubted advantage of our approach is the possibility of obtaining a large amount of readily available and relatively cheap primary material (SN) from wild-type mice, which can be used to solve both research and applied problems. In addition, this method can be easily adapted to the isolation of DNs from the SN in other animal species, including non-human primates.</p

Image_3_Isolation of living dopaminergic neurons labeled with a fluorescent ligand of the dopamine transporter from mouse substantia nigra as a new tool for basic and applied research.JPEG

Dopaminergic neurons (DNs) of the nigrostriatal system control the motor function, and their degeneration leads to the development of Parkinson’s disease (PD). A stumbling block in the study of DNs in the whole substantia nigra (SN) is the lack of tools to analyze the expression of most of the genes involved in neurotransmission, neurodegeneration, and neuroplasticity, since they are also expressed in other cells of the SN. Therefore, this study aimed to develop a fluorescence-activated cell sorting method for isolating living DNs from the SN of wild-type mice using two fluorescent dyes, DRAQ5 (nuclear stain) and a dopamine uptake inhibitor GBR 12909 coupled to a fluorophore (DN stain). We have developed a method for selecting a population of DNs from the SN of mice, as evidenced by: (i) immunopositivity of 95% of the sorted cells for tyrosine hydroxylase, the first enzyme of dopamine synthesis; (ii) the sorted cells expressing the genes for specific proteins of the dopaminergic phenotype, tyrosine hydroxylase, the dopamine transporter, and vesicular monoamine transporter 2 and non-specific proteins, such as aromatic L-amino acid decarboxylase, non-specific enzyme of dopamine synthesis. We then compared the changes in gene expression found in the sorted DNs and in the SN homogenate in a PD model we developed, reproduced in mice by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Using quantitative PCR, we obtained evidence of the same changes in the expression of specific genes in the sorted DNs of SN and in the SN homogenate of a MPTP mouse model of PD, compared with the control. The undoubted advantage of our approach is the possibility of obtaining a large amount of readily available and relatively cheap primary material (SN) from wild-type mice, which can be used to solve both research and applied problems. In addition, this method can be easily adapted to the isolation of DNs from the SN in other animal species, including non-human primates.</p

Image_5_Isolation of living dopaminergic neurons labeled with a fluorescent ligand of the dopamine transporter from mouse substantia nigra as a new tool for basic and applied research.JPEG

Dopaminergic neurons (DNs) of the nigrostriatal system control the motor function, and their degeneration leads to the development of Parkinson’s disease (PD). A stumbling block in the study of DNs in the whole substantia nigra (SN) is the lack of tools to analyze the expression of most of the genes involved in neurotransmission, neurodegeneration, and neuroplasticity, since they are also expressed in other cells of the SN. Therefore, this study aimed to develop a fluorescence-activated cell sorting method for isolating living DNs from the SN of wild-type mice using two fluorescent dyes, DRAQ5 (nuclear stain) and a dopamine uptake inhibitor GBR 12909 coupled to a fluorophore (DN stain). We have developed a method for selecting a population of DNs from the SN of mice, as evidenced by: (i) immunopositivity of 95% of the sorted cells for tyrosine hydroxylase, the first enzyme of dopamine synthesis; (ii) the sorted cells expressing the genes for specific proteins of the dopaminergic phenotype, tyrosine hydroxylase, the dopamine transporter, and vesicular monoamine transporter 2 and non-specific proteins, such as aromatic L-amino acid decarboxylase, non-specific enzyme of dopamine synthesis. We then compared the changes in gene expression found in the sorted DNs and in the SN homogenate in a PD model we developed, reproduced in mice by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Using quantitative PCR, we obtained evidence of the same changes in the expression of specific genes in the sorted DNs of SN and in the SN homogenate of a MPTP mouse model of PD, compared with the control. The undoubted advantage of our approach is the possibility of obtaining a large amount of readily available and relatively cheap primary material (SN) from wild-type mice, which can be used to solve both research and applied problems. In addition, this method can be easily adapted to the isolation of DNs from the SN in other animal species, including non-human primates.</p

Image_6_Isolation of living dopaminergic neurons labeled with a fluorescent ligand of the dopamine transporter from mouse substantia nigra as a new tool for basic and applied research.JPEG

Dopaminergic neurons (DNs) of the nigrostriatal system control the motor function, and their degeneration leads to the development of Parkinson’s disease (PD). A stumbling block in the study of DNs in the whole substantia nigra (SN) is the lack of tools to analyze the expression of most of the genes involved in neurotransmission, neurodegeneration, and neuroplasticity, since they are also expressed in other cells of the SN. Therefore, this study aimed to develop a fluorescence-activated cell sorting method for isolating living DNs from the SN of wild-type mice using two fluorescent dyes, DRAQ5 (nuclear stain) and a dopamine uptake inhibitor GBR 12909 coupled to a fluorophore (DN stain). We have developed a method for selecting a population of DNs from the SN of mice, as evidenced by: (i) immunopositivity of 95% of the sorted cells for tyrosine hydroxylase, the first enzyme of dopamine synthesis; (ii) the sorted cells expressing the genes for specific proteins of the dopaminergic phenotype, tyrosine hydroxylase, the dopamine transporter, and vesicular monoamine transporter 2 and non-specific proteins, such as aromatic L-amino acid decarboxylase, non-specific enzyme of dopamine synthesis. We then compared the changes in gene expression found in the sorted DNs and in the SN homogenate in a PD model we developed, reproduced in mice by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Using quantitative PCR, we obtained evidence of the same changes in the expression of specific genes in the sorted DNs of SN and in the SN homogenate of a MPTP mouse model of PD, compared with the control. The undoubted advantage of our approach is the possibility of obtaining a large amount of readily available and relatively cheap primary material (SN) from wild-type mice, which can be used to solve both research and applied problems. In addition, this method can be easily adapted to the isolation of DNs from the SN in other animal species, including non-human primates.</p

Image_8_Isolation of living dopaminergic neurons labeled with a fluorescent ligand of the dopamine transporter from mouse substantia nigra as a new tool for basic and applied research.JPEG

Dopaminergic neurons (DNs) of the nigrostriatal system control the motor function, and their degeneration leads to the development of Parkinson’s disease (PD). A stumbling block in the study of DNs in the whole substantia nigra (SN) is the lack of tools to analyze the expression of most of the genes involved in neurotransmission, neurodegeneration, and neuroplasticity, since they are also expressed in other cells of the SN. Therefore, this study aimed to develop a fluorescence-activated cell sorting method for isolating living DNs from the SN of wild-type mice using two fluorescent dyes, DRAQ5 (nuclear stain) and a dopamine uptake inhibitor GBR 12909 coupled to a fluorophore (DN stain). We have developed a method for selecting a population of DNs from the SN of mice, as evidenced by: (i) immunopositivity of 95% of the sorted cells for tyrosine hydroxylase, the first enzyme of dopamine synthesis; (ii) the sorted cells expressing the genes for specific proteins of the dopaminergic phenotype, tyrosine hydroxylase, the dopamine transporter, and vesicular monoamine transporter 2 and non-specific proteins, such as aromatic L-amino acid decarboxylase, non-specific enzyme of dopamine synthesis. We then compared the changes in gene expression found in the sorted DNs and in the SN homogenate in a PD model we developed, reproduced in mice by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Using quantitative PCR, we obtained evidence of the same changes in the expression of specific genes in the sorted DNs of SN and in the SN homogenate of a MPTP mouse model of PD, compared with the control. The undoubted advantage of our approach is the possibility of obtaining a large amount of readily available and relatively cheap primary material (SN) from wild-type mice, which can be used to solve both research and applied problems. In addition, this method can be easily adapted to the isolation of DNs from the SN in other animal species, including non-human primates.</p

Image_2_Isolation of living dopaminergic neurons labeled with a fluorescent ligand of the dopamine transporter from mouse substantia nigra as a new tool for basic and applied research.JPEG

Dopaminergic neurons (DNs) of the nigrostriatal system control the motor function, and their degeneration leads to the development of Parkinson’s disease (PD). A stumbling block in the study of DNs in the whole substantia nigra (SN) is the lack of tools to analyze the expression of most of the genes involved in neurotransmission, neurodegeneration, and neuroplasticity, since they are also expressed in other cells of the SN. Therefore, this study aimed to develop a fluorescence-activated cell sorting method for isolating living DNs from the SN of wild-type mice using two fluorescent dyes, DRAQ5 (nuclear stain) and a dopamine uptake inhibitor GBR 12909 coupled to a fluorophore (DN stain). We have developed a method for selecting a population of DNs from the SN of mice, as evidenced by: (i) immunopositivity of 95% of the sorted cells for tyrosine hydroxylase, the first enzyme of dopamine synthesis; (ii) the sorted cells expressing the genes for specific proteins of the dopaminergic phenotype, tyrosine hydroxylase, the dopamine transporter, and vesicular monoamine transporter 2 and non-specific proteins, such as aromatic L-amino acid decarboxylase, non-specific enzyme of dopamine synthesis. We then compared the changes in gene expression found in the sorted DNs and in the SN homogenate in a PD model we developed, reproduced in mice by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Using quantitative PCR, we obtained evidence of the same changes in the expression of specific genes in the sorted DNs of SN and in the SN homogenate of a MPTP mouse model of PD, compared with the control. The undoubted advantage of our approach is the possibility of obtaining a large amount of readily available and relatively cheap primary material (SN) from wild-type mice, which can be used to solve both research and applied problems. In addition, this method can be easily adapted to the isolation of DNs from the SN in other animal species, including non-human primates.</p

Image_7_Isolation of living dopaminergic neurons labeled with a fluorescent ligand of the dopamine transporter from mouse substantia nigra as a new tool for basic and applied research.JPEG

Dopaminergic neurons (DNs) of the nigrostriatal system control the motor function, and their degeneration leads to the development of Parkinson’s disease (PD). A stumbling block in the study of DNs in the whole substantia nigra (SN) is the lack of tools to analyze the expression of most of the genes involved in neurotransmission, neurodegeneration, and neuroplasticity, since they are also expressed in other cells of the SN. Therefore, this study aimed to develop a fluorescence-activated cell sorting method for isolating living DNs from the SN of wild-type mice using two fluorescent dyes, DRAQ5 (nuclear stain) and a dopamine uptake inhibitor GBR 12909 coupled to a fluorophore (DN stain). We have developed a method for selecting a population of DNs from the SN of mice, as evidenced by: (i) immunopositivity of 95% of the sorted cells for tyrosine hydroxylase, the first enzyme of dopamine synthesis; (ii) the sorted cells expressing the genes for specific proteins of the dopaminergic phenotype, tyrosine hydroxylase, the dopamine transporter, and vesicular monoamine transporter 2 and non-specific proteins, such as aromatic L-amino acid decarboxylase, non-specific enzyme of dopamine synthesis. We then compared the changes in gene expression found in the sorted DNs and in the SN homogenate in a PD model we developed, reproduced in mice by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Using quantitative PCR, we obtained evidence of the same changes in the expression of specific genes in the sorted DNs of SN and in the SN homogenate of a MPTP mouse model of PD, compared with the control. The undoubted advantage of our approach is the possibility of obtaining a large amount of readily available and relatively cheap primary material (SN) from wild-type mice, which can be used to solve both research and applied problems. In addition, this method can be easily adapted to the isolation of DNs from the SN in other animal species, including non-human primates.</p

Image_4_Isolation of living dopaminergic neurons labeled with a fluorescent ligand of the dopamine transporter from mouse substantia nigra as a new tool for basic and applied research.JPEG

Dopaminergic neurons (DNs) of the nigrostriatal system control the motor function, and their degeneration leads to the development of Parkinson’s disease (PD). A stumbling block in the study of DNs in the whole substantia nigra (SN) is the lack of tools to analyze the expression of most of the genes involved in neurotransmission, neurodegeneration, and neuroplasticity, since they are also expressed in other cells of the SN. Therefore, this study aimed to develop a fluorescence-activated cell sorting method for isolating living DNs from the SN of wild-type mice using two fluorescent dyes, DRAQ5 (nuclear stain) and a dopamine uptake inhibitor GBR 12909 coupled to a fluorophore (DN stain). We have developed a method for selecting a population of DNs from the SN of mice, as evidenced by: (i) immunopositivity of 95% of the sorted cells for tyrosine hydroxylase, the first enzyme of dopamine synthesis; (ii) the sorted cells expressing the genes for specific proteins of the dopaminergic phenotype, tyrosine hydroxylase, the dopamine transporter, and vesicular monoamine transporter 2 and non-specific proteins, such as aromatic L-amino acid decarboxylase, non-specific enzyme of dopamine synthesis. We then compared the changes in gene expression found in the sorted DNs and in the SN homogenate in a PD model we developed, reproduced in mice by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Using quantitative PCR, we obtained evidence of the same changes in the expression of specific genes in the sorted DNs of SN and in the SN homogenate of a MPTP mouse model of PD, compared with the control. The undoubted advantage of our approach is the possibility of obtaining a large amount of readily available and relatively cheap primary material (SN) from wild-type mice, which can be used to solve both research and applied problems. In addition, this method can be easily adapted to the isolation of DNs from the SN in other animal species, including non-human primates.</p

Targeting ErbB3 Receptor in Cancer with Inhibitory Antibodies from Llama

The human ErbB3 receptor confers resistance to the pharmacological inhibition of EGFR and HER2 receptor tyrosine kinases in cancer, which makes it an important therapeutic target. Several anti-ErbB3 monoclonal antibodies that are currently being developed are all classical immunoglobulins. We took a different approach and discovered a group of novel heavy-chain antibodies targeting the extracellular domain of ErbB3 via a phage display of an antibody library from immunized llamas. We first produced three selected single-domain antibodies, named BCD090-P1, BCD090-M2, and BCD090-M456, in E. coli, as SUMO fusions that yielded up to 180 mg of recombinant protein per liter of culture. Then, we studied folding, aggregation, and disulfide bond formation, and showed their ultimate stability with half-denaturation of the strongest candidate, BCD090-P1, occurring in 8 M of urea. In surface plasmon resonance experiments, two most potent antibodies, BCD090-P1 and BCD090-M2, bound the extracellular domain of ErbB3 with 1.6 nM and 15 nM affinities for the monovalent interaction, respectively. The receptor binding was demonstrated by immunofluorescent confocal microscopy on four different ErbB3+ cancer cell lines. We observed that BCD090-P1 and BCD090-M2 bind noncompetitively to two distinct epitopes on the receptor. Both antibodies inhibited the ErbB3-driven proliferation of MCF-7 breast adenocarcinoma cells and HER2-overexpressing SK-BR-3 cells, with the EC50 in the range of 0.1–25 μg/mL. BCD090-M2 directly blocks ligand binding, whereas BCD090-P1 does not compete with the ligand and presumably acts through a distinct allosteric mechanism. We anticipate that these llama antibodies can be used to engineer new biparatopic anti-ErbB3 or bispecific anti-ErbB2/3 antibodies