Take a look at the Recent articles

Peptidoglycan aptamers biodistribution in infection-bearing mice

Iêda Mendes Ferreira

Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Rua Professor Mário Werneck S/N°, Cidade Universitária-Campus da UFMG, 31120-970, Belo Horizonte, MG – Brasil

E-mail : bhuvaneswari.bibleraaj@uhsm.nhs.uk

Camila Maria de Sousa Lacerda

Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Rua Professor Mário Werneck S/N°, Cidade Universitária-Campus da UFMG, 31120-970, Belo Horizonte, MG – Brasil

Sara Roberta dos Santos

Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Rua Professor Mário Werneck S/N°, Cidade Universitária-Campus da UFMG, 31120-970, Belo Horizonte, MG – Brasil

André Luís Branco de Barros

Departamento de Análises Clínicas e Toxicológicas - Faculdade de Farmácia, Universidade Federal de Minas Gerais (UFMG), Cidade Universitária – Campus da UFMG, 31270-091, Belo Horizonte, MG – Brasil

Simone Odília Fernandes

Departamento de Análises Clínicas e Toxicológicas - Faculdade de Farmácia, Universidade Federal de Minas Gerais (UFMG), Cidade Universitária – Campus da UFMG, 31270-091, Belo Horizonte, MG – Brasil

Valbert Nascimento Cardoso

Departamento de Análises Clínicas e Toxicológicas - Faculdade de Farmácia, Universidade Federal de Minas Gerais (UFMG), Cidade Universitária – Campus da UFMG, 31270-091, Belo Horizonte, MG – Brasil

Antero Silva Ribeiro de Andrade

Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Rua Professor Mário Werneck S/N°, Cidade Universitária-Campus da UFMG, 31120-970, Belo Horizonte, MG – Brasil

DOI: 10.15761/NMBI.1000137

Article
Article Info
Author Info
Figures & Data

Abstract

Introduction: Acid Nucleic aptamers are short single-stranded oligonucleotides that display high affinity and selectivity for a given target. Aptamers contain many features that are advantageous for radiopharmaceuticals development. Peptidoglycan is a cell wall polymer common to both Gram-positive and Gram-negative bacteria. In the present study, the potential of two peptidoglycan aptamers for bacterial infection foci identification was evaluated.

Material and methods: The peptidoglycan aptamers were labeled with 99mTc by the direct method and the stability of each 99mTc-aptamer complex was evaluated in saline, plasma and in presence of cysteine. The aptamers degradation by plasma nucleases was also assessed. Bacterial‑infected (Staphylococcus aureus) mice and fungal-infected mice (Candida albicans) were used for the ex vivo biodistribution studies with the 99mTc-aptamers.

Results and discussion: The aptamers were not degraded by plasma nucleases. High radiolabel yields were obtained by the direct method and the complexes were stable in presence of saline and plasma. Some trans chelation was observed in the presence of cysteine. The 99mTc-pepdigoglycan aptamers uptake in the bacterial infection foci were significantly higher than the control (a radiolabeled oligonucleotide library) and their uptake in the fungal infection model.

Conclusion: Both radiolabeled peptidoglycan aptamers present specific uptake in the bacterial infection foci highlighting the potential of these molecules as radiotracer for bacterial infection.

Key words

Aptamers, peptidoglycan, technetium-99m, biodistribution, bacterial infection

Introduction

Nuclear imaging of bacterial infections has been a developing field for more than 50 years. In the last decade, significant efforts have been made to develop bacteria-specific radiolabeled tracers that bind or accumulate only in the bacterial cells differentiating bacterial infection from inflammation and from other types of infection. The most used imaging agents as 18F-fluorodeoxyglucose (18F-FDG), 67Ga-citrate, and in vitro-radiolabeled leukocytes are not specific for infection, targeting the infection associated inflammation, and so not solving a frequent clinical problem that is differentiating active infection from other causes of inflammation [1].

Acid Nucleic aptamers are short single-stranded oligonucleotides that display high affinity and selectivity for a given target. Aptamers contain many features that are advantageous for radiopharmaceuticals development. They seem to be non-toxic and non-immunogenic, have small size (10 to 20 kDa) and fast clearance, allowing superior target-to-noise ratios at early time points. Since their discovery, several aptamers have been used as targeting molecule of radiopharmaceuticals in preclinical studies. The majority has been radiolabeled with 99mTc for SPECT (single-photon emission computed tomography) imaging of cancer-related targets [2].  Our research group has explored radiolabeled aptamers for infection diagnosis [3-6].  Radiolabeled aptamers specific for infectious agents could give an important contribution to infections diagnosis through scintigraphy, allowing distinguishing between infection and inflammation and identifying the microorganism causing the infection. Pathogen-specific imaging techniques could avoid the inappropriate use of antibiotics for noninfectious entities or for non-bacterial infections and provide a way to monitor antibiotic treatments.

Peptidoglycan is a cell wall polymer common to both Gram-positive and Gram-negative bacteria that coats the entire cell.  In the peptidoglycan structure, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) alternate to form the carbohydrate polymer that is connected by multiple peptide cross-links [7]. The particular composition of peptidoglycan makes it a possible target for specific bacterial recognition. In a previous work, Ferreira et al. (2014) [8] selected two aptamers for peptidoglycan. The radiolabeled aptamers showed high binding capacity for S. aureus and E. coli cells in vitro, but the binding to C. albicans and human fibroblasts was negligible. Graziani et al.  [9] demonstrated that the both peptidoglycan aptamers bind with high efficiency to all Gram‑positive and Gram-negative species tested. In the present study, the peptidoglycan aptamers potential for bacterial infection foci identification was evaluated by ex vivo biodistribution studies. Two different experimental infection models were used: Bacterial-infected mice (S. aureus) and fungal-infected mice (C. albicans).

Material and Methods

Microorganisms and culture

Staphylococcus aureus (ATCC 25923) and Candida albicans (ATCC 18804) were cultured on BHI solid (HI media Laboratories Pvt Ltd.) in Petri dishes at 37 °C and sub-cultured every seven days.

Animals

The mice were kept in cages with wood shavings, water and common food (ad libitum) in ordinary shelves. The Swiss mice infected with C. albicans were firstly immunosuppressed by gamma radiation in a uniform 60Co source at the Gamma Irradiation Laboratory of the Center of Nuclear Technology Development (CDTN, Brazil). A dose of 2.5 Gray and a dose rate of 75 Gray/h were used. After irradiation, the animals were maintained in autoclaved cages with wood shavings, water and food (ad libitum). All protocols were approved by the local Ethics Committee for Animal Experimentation of the Federal University of Minas Gerais (CETEA / UFMG), Protocol n° 108/2014.

Chemicals

The 99mTc was obtained from a molybdenum generator (IPEN/Brazil). Reagents, including tricine, ethylenediamine-N´, N´-diacetic acid (EDDA), SnCl2.2H2O were purchased from Sigma-Aldrich (São Paulo, Brazil). The aptamers
Antibac1 (5’TCGCGCGAGTCGTCTGGGGACAGGGAGTGCGCTGCTCCCCCCGCACGTCCTCCC 3’) and
Antibac2 (5’TCGCGCGAGTCGTCTGGGGGACTAGAGGACTTGTGCGGCCCCGCATCGTCCTCCC3'),
previously selected by Ferreira et al. (2014), were synthesized by Integrated DNA Technologies (IDT) with the introduction at the 3´end of an amino group with a 6 carbons spacer and at the 5' end of an inverted thymidine.

Evaluation of aptamer degradation by plasma nucleases

Blood (3 mL) was collected from Swiss mice and the plasma fraction was separated by centrifugation (700g). EDTA (0.1M) was used as anticoagulant. The aptamers Antibac1 and Antibac2 modified at the 3' and 5' ends were incubated separately with the plasma at 37 °C in the following ratio: 1 μL of aptamer solution (200 pmol / μL) for 9 μl of plasma. Aliquots of 10 μl were removed 5 m, 1 h, 3 h, 6 h and 24 h after and submitted to electrophoresis on 2% agarose gel stained with ethidium bromide.

Aptamer radiolabeling with 99mTc and radiolabel yield determination

Labeling with 99mTc was performed by the direct method according to Correa et al (2014) [10].  For the labeling reaction 111.6 mol of tricine and 28.3 mol of EDDA were added to 300 µL of 0.9% saline. Then, 10 µL of aptamer (200 pmol/µL) followed by 100 µL of SnCl2·2H2O (8.9 mM in HCl 0.25 N) were added to the solution. The pH was adjusted to 7.0 with 1.0 N NaOH. The bottle was sealed, and vacuum was applied with a syringe. The activity of 296 MBq of a 99mTc‑pertechnetate solution (Na99m TcO4-) was added. Then, the solution was boiled in water bath for 15 min and next cooled in running water.  The injected activity for each animal was 14.8 MBq. An oligonucleotide DNA library (random sequences) was labeled in the same way and used as control.

The radiolabel yield of 99mTc-aptamer complex was assessed by ascending instant thin-layer chromatography (TLC) using silica gel-coated fiberglass sheets and two solvent systems: (1) 100% acetone to determine the percentage of TcO4- and (2) 0.9% NaCl solution with 5% NH4OH to determine the percentage of TcO2. The labeled product (99mTc-aptamer) remained at the point of application when 100% acetone was used as the mobile phase (Rf=0) and the labeled product moved with the solvent front when 0.9% NaCl solution with 5% NH4OH was used as the mobile phase (Rf=1). The radiolabel yield was determined according to the following equation: Labeling percentage = 100 – (% TcO4- + % TcO2).

Stability of 99mTc labeled aptamers

The stability of each 99mTc-aptamer complex was evaluated in saline, plasma and cysteine excess (50 mol of cysteine per mol of aptamer) by TLC. Analysis of stability was performed by adding 100 µL of the radiolabeled aptamers solution in tubes containing 1.1 mL of 0.9% NaCl, mice plasma or cysteine solution. The solutions of saline and cysteine were stored at room temperature and the plasma was incubated at 37 °C. All solutions were analyzed at 5 min, 1 h, 3 h, and 6 h later by TLC.

Biodistribution

The animals were anesthetized with a mixture of xylazine (15 mg / kg) and ketamine (80 mg / kg). The mice were infected intramuscularly in the right thigh with 1 x 106 cells of S. aureus (ATCC 25923) suspended in 100 µL of saline or infected in the same way with 1 x 105 cells of C. albicans (ATCC 18804). Groups of Swiss mice (20-25 g weight) containing 6 animals each (n = 6) were used. The animals infected with C. albicans were immunosuppressed before infection as described earlier. A visible swelling was observed on the infected thigh of all animals at 24 h after the intervention. So, 100 µL (14.8 MBq) of the radiolabeled aptamer solution or the radiolabeled oligonucleotide library (control) were injected by the tail vein in each animal. The mice were euthanized at 3 h post-injection and tissue samples (blood, liver, spleen, stomach, heart, lung, kidney, infected thigh muscle, and non-infected thigh muscle) were dissected, weighed, and their activities measured in a gamma counter. At the time of euthanasia, samples from infection foci were obtained for microbial culture and only the animals that tested positive were considered in this study. The results were expressed as the percentage of injected dose per gram of tissue (%ID/g). Target/non‑target ratios were obtained from the analysis of radiation measured in the right thigh infected muscle in relation to radiation measured in the left thigh muscle.

Statistical analysis

All data were expressed as mean ± SD and analyzed by GraphPad PRISM version 5.01 software. The analysis of variance (ANOVA) with a confidence interval of 95% and Tukey multiple comparison test were used. A P value <0.05 was considered to indicate a statistically significant difference.

Results and Discussion

The figure 1 shows the results of the assay of aptamers degradation by plasma nucleases.  The aptamers Antibac1 and Antibac2, modified with the introduction at the 3´end of an amino group with a 6 carbons spacer and at the 5' end of an inverted thymidine, were not degraded by plasma nucleases and remained stable up to 24 h. This stability in plasma is an important property for a radiopharmaceutical candidate.

Figure 1. Evaluation of Antibac1 and Antibac2 degradation by plasma nucleases.

The aptamers Antibac1 and Antibac2 modified at the 3' and 5' ends were incubated separately with the plasma at 37 °C. Aliquots of 10 μl were removed 5 m (A), 1 h (B), 3 h (C), 6 h (D) and 24 h (E) after and submitted to electrophoresis on 2% agarose gel stained with ethidium bromide. (1) DNA Ladder of 50 pb, (2) Antibac1 and (3) Antibac2

The aptamers were labeled with 99mTc by the direct method developed by Correa et al (2014), which allows high radiolabel yields. In the present study, only radiotracer preparations presenting radiolabel yields higher than 90% were used in the experiments.  The stability of each 99mTc-aptamer complex was evaluated in saline, plasma and in presence of cysteine. A high complex stability was verified in presence of saline and plasma for both aptamers complexes, since the percentage of radiolabeling was kept above 90% up to 6 h. Some trans chelation was observed in the presence of cysteine (50 mol of cysteine per mol of aptamer). The radiolabeled yields percentages in cysteine excess for Antibac1 and Antibac2 were 73.1 ± 0.1 and 81.9 ± 1.9 at 3 h, respectively, and after 6 h, these values were 71.9 ± 0.6 and 73.8 ± 1.6 (Tables 1 and 2). Based on these findings it was concluded that the 99mTc‑aptamers complexes were suitable for further in vivo assays.

Table 1. In vitro stability of the 99mTc‑Antibac1 complex in the presence of 0.9% saline, plasma, and molar excess of cysteine (% radiolabeled yield).

Time

Saline

Plasma

Cys 50:1

5 min

95.50 ± 1.07

93.87 ± 0.21

96.09 ± 0.14

1 h

92.49 ± 2.09

97.49 ± 0.09

72.48 ± 1.00

3 h

92.93 ± 0.19

97.81 ± 0.04

73.09 ± 1.00

6 h

87.00 ± 0.43

97.34 ± 0.26

71.89 ± 0.61

Cys- Cysteine

Table 2.  In vitro stability of the 99mTc‑Antibac2 complex in the presence of 0.9% saline, plasma, and a molar excess of cysteine (% radiolabeled yield).

Time

Saline

Plasma

Cys 50:1

5 min

95.46 ± 1.08

95.70 ± 0.74

89.39 ± 1.24

1 h

91.96 ± 0.28

97.62 ± 0.09

89.71 ± 1.26

3 h

90.01 ± 0.51

98.06 ± 0.74

81.94 ± 1.86

6 h

91.25 ± 0.96

97.01 ± 1.46

73.81 ± 1.62

Cys- Cysteine

The biodistribution studies were carried out in two different experimental infection models: Bacterial‑infected mice (S. aureus) and fungal-infected mice (C. albicans). The fungal-infected mice group was used to confirm the aptamers specificity for the bacterial infection foci. This control group was also useful to measure the radiotracer uptake due to the local inflammatory process that accompanies an infection, producing vasodilatation and increased capillary permeability. Many radiopharmaceuticals used for infection diagnosis actually accumulate in the infection site mainly due to these vascular effects and they are not specific to infection [Ferro-Flores et al., 2012]. To help the evaluation of this nonspecific uptake, a 99mTc‑radiolabeled library consisting of oligonucleotides with random sequences was also used as a control in both infections models. These oligonucleotides without specificity for the microbial targets act as blood flow markers and non-specific uptake indicators.

Figure 2 presents the biodistribution for 99mTc-Antibac1, 99mTc-Antibac2 and the 99mTc‑library in the S. aureus-infected mice. The results showed a higher uptake of the 99mTc-Antibac1 and 99mTc‑Antibac2 in the infected thigh compared to the radiation measured in the left thigh muscle. The target to non-target (T/NT) ratio for 99mTc-Antibac1and 99mTc-Antibac2 were of 3.2 ± 0.2 and 2.6 ± 0.7, respectively. These ratios were statistically higher (p < 0.05) than that found for the 99mTc‑library (1.5 ± 0.1).

Figure 2. Biodistribution in the bacterial‑infected model.

The Antibac1, Antibac2 and the library were labeled with 99mTc and injected into the tail vein of S. aureus infected mice. The mice were euthanized at 3 h after injection, tissue samples were dissected, and their activities were measured in a gamma counter. The symbol (*) indicates a statistical difference in the radiation uptake between the infected right thigh and the uninfected left thigh (p < 0.05).

The biodistribution in the fungal-infected model is shown in Figure 3. No statistical difference (p ˃ 0.05) was observed between 99mTc‑Antibac1, 99mTc-Antibac2 and 99mTc‑library uptake. The T/NT ratios were of 1.5 ± 0.1, 1.7 ± 0.2 and 1.5 ± 0.2, respectively, indicating non-specificity of peptidoglycan aptamers to C. albicans infection foci.

Figure 3. Biodistribution in the fungal-infected model.

The Antibac1, Antibac2 and the library were labeled with 99mTc and injected into the tail vein of Candida albicans infected mice. The mice were euthanized at 3 h after injection, tissue samples were dissected, and their activities were measured in a gamma counter.

All biodistribution studies showed a high percentage of radioactivity in the kidneys. This finding indicates a main renal excretion rout, which is consistent with the hydrophilic nature and small size of aptamers. Because chelating agents were not used in the labeling process, the biodistribution profile seemed to reflect mainly the aptamer properties.

The uptake of the 99mTc‑library in the infection foci can be correlated to the increased capillary permeability and vasodilatation triggered by the inflammation associated to the infection, since T/NT ratios due the 99mTc‑library were similar in both infection models (1.5). These ratios were also comparable to the T/NT ratios of 99mTc‑Antibac1 and 99mTc-Antibac2 in the fungal infection model, in which the aptamers have no affinity for the microorganism causing the infection. By the other side 99mTc‑Antibac1 and 99mTc‑Antibac2 allowed high T/NT in the bacterial infection model, statistically higher than found for the 99mTc‑library and the T/NT ratios verified for these aptamers in the fungal infection model.

Aptamers are molecules that provide high specificity for pre-selected targets. In this work, we evaluate aptamers for the peptidoglycan, present in the cell walls of all bacterial species, and therefore they work as generic probes for bacteria identification. However, aptamers can also be used for scintigraphy identification of a particular bacterial specie [5], highlighting the potential of these molecules for the development of radiopharmaceuticals for infection diagnosis.

Conclusions

Both peptidoglycan aptamers were successfully labeled with 99mTc, showing stability for in vivo studies. By using two different infection models and a control based on a radiolabeled oligonucleotide library was possible to demonstrate that both peptidoglycan aptamers present specific uptake in the bacterial infection foci. 

Conflict of interest

None

Acknowledgments

This research was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) (TEC-APQ-02247-16).

References

  1. Ferro Flores G, Ocampo Garcia BE, Melendez Alafort L (2012) Development of  specific radiopharmaceuticals for infection imaging by targeting infectious micro-organisms. Curr Pharm Des 18: 1098-1106. [Crossref]
  2. Gijs M, Aertsa A, Impens N, Baatout S, Luxen A (2016) Aptamers as radiopharmaceuticals for nuclear imaging and therapy. Nucl Med Biol 43: 253-271.  [Crossref]
  3. Santos SR, Corrêa CR, De Barros ALB, Serakides R, Fernandes SO et al. (2015) Identification of Staphylococcus aureus infection by aptamers directly radiolabeled with technetium-99 m. Nucl Med Biol 42: 292-298. [Crossref]
  4. de Sousa Lacerda CM, Ferreira IM, Dos Santos SR, de Barros AL, Fernandes SO, et al. (2017) (1→3)-β-D-glucan aptamers labeled with technetium-99m: Biodistribution and imaging in experimental models of bacterial and fungal infection. Nucl Med Biol 46: 19-24. [Crossref]
  5. Santos SRD, de Sousa Lacerda CM, Ferreira IM, de Barros ALB, Fernandes SO, et al. (2017)  Scintigraphic imaging of Staphylococcus aureus infection using 99mTc radiolabeled aptamers. Appl Radiat Isot 128: 22-27. [Crossref]
  6. Ferreira IM, de Sousa Lacerda CM, Dos Santos SR, de Barros ALB, Fernandes SO, et al. (2017) Detection of bacterial infection by a technetium-99m-labeled peptidoglycan aptamer. Biomed Pharmacother 93: 931-938. [Crossref]
  7. Silhavy TJ, Kahne D, Walker S (2010) The bacterial cell envelope. Cold Spring Harb Perspect Biol 2: a000414. [Crossref]
  8. Ferreira IM, de Sousa Lacerda CM, Faria LS, Corrêa CR, Andrade ASR (2014) Selection of Peptidoglycan-Specific Aptamers for Bacterial Cells Identification. Appl Biochem Biotechnol 174: 2548-2556. [Crossref]
  9. Graziani AC, Stets MI, Lopes AL,Schluga PH, Marton S, et al. (2017) High efficiency binding aptamers for a wide range of sepsis bacterial agents. J Microbiol Biotechnol 27: 838-843. [Crossref]
  10. Correa CR, Barros ALB, Ferreira CA, Goes AM, Cardoso VN, et al. (2014) Aptamers directly radiolabeled with technetium-99m as a potential agent capable of identifying carcinoembryonic antigen (CEA) in tumor cells T84. Bioorg Med Chem Lett 24: 1998-2001. [Crossref]

Editorial Information

Editor-in-Chief

Dr. Rex Cheung
Columbia University School of Mediciness

Article Type

Research Article

Publication history

Received date: April 16, 2018
Accepted date: April 26, 2018
Published date: May 01, 2018

Copyright

© 2018 Ferreira IM. This is an open‑access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation

Ferreira IM (2018) Peptidoglycan aptamers biodistribution in infection-bearing mice. Nucl Med Biomed Imaging 3: DOI: 10.15761/NMBI.1000137

Corresponding author

Antero Silva Ribeiro de Andrade

Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Rua Professor Mário Werneck S/N°, Cidade Universitária-Campus da UFMG, 31120-970, Belo Horizonte, MG – Brasil

E-mail : bhuvaneswari.bibleraaj@uhsm.nhs.uk

Figure 1. Evaluation of Antibac1 and Antibac2 degradation by plasma nucleases.

The aptamers Antibac1 and Antibac2 modified at the 3' and 5' ends were incubated separately with the plasma at 37 °C. Aliquots of 10 μl were removed 5 m (A), 1 h (B), 3 h (C), 6 h (D) and 24 h (E) after and submitted to electrophoresis on 2% agarose gel stained with ethidium bromide. (1) DNA Ladder of 50 pb, (2) Antibac1 and (3) Antibac2

Figure 2. Biodistribution in the bacterial‑infected model.

The Antibac1, Antibac2 and the library were labeled with 99mTc and injected into the tail vein of S. aureus infected mice. The mice were euthanized at 3 h after injection, tissue samples were dissected, and their activities were measured in a gamma counter. The symbol (*) indicates a statistical difference in the radiation uptake between the infected right thigh and the uninfected left thigh (p < 0.05).

Figure 3. Biodistribution in the fungal-infected model.

The Antibac1, Antibac2 and the library were labeled with 99mTc and injected into the tail vein of Candida albicans infected mice. The mice were euthanized at 3 h after injection, tissue samples were dissected, and their activities were measured in a gamma counter.

Table 1. In vitro stability of the 99mTc‑Antibac1 complex in the presence of 0.9% saline, plasma, and molar excess of cysteine (% radiolabeled yield).

Time

Saline

Plasma

Cys 50:1

5 min

95.50 ± 1.07

93.87 ± 0.21

96.09 ± 0.14

1 h

92.49 ± 2.09

97.49 ± 0.09

72.48 ± 1.00

3 h

92.93 ± 0.19

97.81 ± 0.04

73.09 ± 1.00

6 h

87.00 ± 0.43

97.34 ± 0.26

71.89 ± 0.61

Cys- Cysteine

Table 2.  In vitro stability of the 99mTc‑Antibac2 complex in the presence of 0.9% saline, plasma, and a molar excess of cysteine (% radiolabeled yield).

Time

Saline

Plasma

Cys 50:1

5 min

95.46 ± 1.08

95.70 ± 0.74

89.39 ± 1.24

1 h

91.96 ± 0.28

97.62 ± 0.09

89.71 ± 1.26

3 h

90.01 ± 0.51

98.06 ± 0.74

81.94 ± 1.86

6 h

91.25 ± 0.96

97.01 ± 1.46

73.81 ± 1.62

Cys- Cysteine