Broad Spectrum Proteomics Analysis of the Inferior Colliculus Following Acute

bioRxiv preprint doi: https://doi.org/10.1101/237370; this version posted December 28, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Running Title: Broad Spectrum Proteomics Analysis of the Inferior Colliculus following Acute

2 Hydrogen Sulfide Exposure

3 Broad Spectrum Proteomics Analysis of the Inferior

4 Colliculus following Acute Hydrogen Sulfide Exposure

5 Dong-Suk Kim1, Poojya Anantharam1a, Andrea Hoffmann2, Mitchell L. Meade3, Nadja Grobe3,

6 Jeffery M. Gearhart2, Elizabeth M. Whitley4, Belinda Mahama1b, Wilson K. Rumbeiha1*

7 1Veterinary Diagnostic & Production Animal Medicine, Iowa State University, Ames, IA, US

8 2Henry M Jackson Foundation on contract 711HPW/USAFSAM/FHOF, Wright Patterson Air

9 Force Base, Dayton, OH, US

10 3711HPW/RHDJ, Wright Patterson Air Force Base, Dayton, OH, US

11 4Pathogenesis, LLC, Gainesville, FL, US

12 *Corresponding Author’s Contact Information:[email protected], 515-294-0630

13 Key Words: Hydrogen Sulfide, Brain Injury, Proteomic Profiling, Proteomic Analysis, TMT

14 labeled LC-MS/MS, Neurodegeneration

15 Present addresses:

16 aPoojya Anantharam: Medical Countermeasures, MRI Global, Kansas City, MO, US

17 bBelinda Mahama: Neuroscience, Brown University, Rhode Island, Providence, US

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18 Abstract: Acute exposure to high concentrations of H2S causes severe brain injury and long-term

19 neurological disorders. The mechanisms of H2S-induced neurodegeneration are not known. To

20 better understand the cellular and molecular mechanisms of H2S-induced neurodegeneration we

21 used a broad-spectrum proteomic analysis approach to search for key molecules in H2S-induced

22 neurotoxicity. Mice were subjected to acute whole body exposure of up to750 ppm of H2S. The

23 H2S-treated group showed behavioral motor deficits and developed severe lesions in the inferior

24 colliculus (IC), part of the brainstem. The IC was microdissected for proteomic analysis. Tandem

25 mass tags (TMT) liquid chromatography mass spectrometry (LC-MS/MS)-based quantitative

26 proteomics was applied for protein identification and quantitation. LC-MS/MS was able to

27 identify 598, 562, and 546 altered proteomic changes for day 1 (2 h post H2S exposure), day 2,

28 and day 4 of H2S exposure, respectively. Mass spectrometry data were analyzed by Perseus

29 1.5.5.3 statistical analysis, and gene ontology heat map clustering. Quantitative real-time PCR

30 was used to confirm some of the H2S-dependent proteomics changes. Taken together, acute

31 exposure to H2S induced behavioral motor deficits along with progressive neurodegeneration

32 including disruption of several biological processes in the IC such as cellular morphology,

33 energy metabolism, and calcium signaling. The obtained broad-spectrum proteomics data may

34 provide important clues to elucidate mechanisms of H2S-induced neurotoxicity.

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36 Highlights:

37  Mice exposed to H2S recapitulated H2S-induced neurotoxicity manifested in humans.

38  The IC in the mouse brain is the most sensitive to H2S-induced neurodegeneration.

39  Proteomic expressions of key proteins were validated at transcription level.

40  Several biological pathways were dysregulated by H2S exposure.

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43 1. Introduction

44 Hydrogen sulfide (H2S) is a highly neurotoxic colorless gas with a “rotten egg” odor (Chou et

45 al., 2016). It is as an environmental pollutant that causes occupational hazards in a variety of

46 industrial processes including the oil and gas industry, intensive animal farming operations,

47 sewer and waste water treatment plants, pulp and paper plants, and gas storage facilities, among

48 several others (Chou et al., 2016). It has been estimated that there are more than 1,000 reports of

49 human exposures to H2S each year in the United States (Chou et al., 2016). Besides accidental

50 H2S poisoning in industrial settings, intentional acute exposure to high concentrations of H2S as

51 a means of suicide has been increasingly observed in Western and Asian societies (Morii et al.,

52 2010; Reedy et al., 2011). The raw chemical ingredients used to generate H2S in such

53 circumstances are readily accessible in local stores (Morii et al., 2010). More significantly, H2S

54 is listed as a high priority chemical by the US Department of Homeland Security because of its

55 potential to be misused in nefarious acts such as chemical terrorism, particularly in confined

56 spaces such as the underground transit systems. Although H2S is toxic at high concentrations, it

57 is also beneficial at physiologic concentrations. For this reason, there is also tremendous interest

58 in potential therapeutic applications of H2S for treatment of a number of human disease

59 conditions. Proposed pharmacological uses of H2S include ulcer treatment, ischemic reperfusion

60 injury, as an anti-inflammatory for treatment of Crohn’s disease, endotoxin induced

61 inflammation, arterial hypertrophy, visceral pain, Parkinson’s disease and cancer among others

62 (Szabo, 2007; Popov, 2013). Furthermore, H2S was shown to activate nitric oxide (NO) synthesis

63 through induction of endothelial nitric oxide synthase. Similar to NO, H2S serves as a

64 gasotransmitter and signaling molecule in the CNS and regulating vasodilation (Tan et al., 2010).

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65 Like most toxicants, the toxicity of H2S exposure is dose-dependent, but H2S characteristically

66 has a steep-dose response curve (Guidotti, 1996). At concentrations below 30 parts per million

67 (ppm), H2S causes headache, coughing and throat irritation. At 150 ppm H2S induces olfactory

68 fatigue and temporary loss of smell after 5-60 min of exposure (Wasch et al., 1989).

69 Concentrations higher than 500 ppm cause headache, dizziness, unconsciousness, and respiratory

70 failure. At high concentration above 1,000 ppm H2S can lead to immediate loss of consciousness,

71 commonly called “knock-down”, and ultimately seizures, and sudden death (Chou et al., 2016).

72 Typically, acute exposure to high concentrations of H2S is associated with high mortality withing

73 a few hours post-exposure (McCabe and Clayton, 1952). Timely rescue of victims can prevent

74 disaster and allow victims to fully recover. Currently, there is no effective antidote to treat

75 victims of H2S gas exposure and the mortality rate remains high (Lindenmann et al.; Herbert,

76 1989; Vicas, 1989; Smith, 1997; Lindenmann et al., 2010). In addition, delayed neurological

77 disorders, which can last for many years, are commonly reported in survivors of acute H2S

78 exposures (Matsuo et al., 1979; Parra et al., 1991; Tvedt et al., 1991a; Tvedt et al., 1991b;

79 Snyder et al., 1995; Kilburn, 2003; Woodall et al., 2005). These delayed neurological sequelae

80 are incapacitating, and require prolonged medical attention that lacks defined medical

81 interventions.

82 The development of effective therapeutics requires a good understanding of the molecular

83 mechanisms and pathways of H2S-induced neurodegeneration and neurological sequelae. These

84 mechanisms remain largely unknown. There is an acute need for countermeasures for treatment

85 of mass civilian casualties of acute H2S poisoning in the field, such as following catastrophic

86 industrial meltdowns or intentional terrorist activities. Elucidating molecular mechanisms

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87 underlying H2S-induced neurotoxicity is essential in developing suitable targeted therapeutics to

88 counter both acute and delayed neurotoxic effects of H2S poisoning in humans.

89 We recently developed a relevant inhalational animal model that exhibits the clinical,

90 pathological, and motor behavioral symptoms of H2S exposed survivors (Anantharam et al.,

91 2017). The objective of this study was to build on our previous work and investigate proteomic

92 changes and altered gene expression in a mouse model of acute H2S-exposure to identify novel

93 toxic mechanisms. In prior studies, we discovered that the central inferior colliculus (IC) region

94 of the brainstem is the most sensitive brain region to H2S-induced neurodegeneration

95 (Anantharam et al., 2017). Consequently, we focused on the IC in this proteomic study, although

96 we have also observed histopathological changes in other parts of the brain such as the thalamus

97 and cortex (Anantharam et al., 2017). This study demonstrated, for the first time, that H2S

98 exposure induces significant proteomic changes in the IC, which may play an important role in

99 execution of H2S-induced neurotoxicity.

100 2. Materials and Methods

101 2.1 Chemicals

102 Hydrogen sulfide gas was purchased from Airgas (Radnor, PA). RNeasy mini kit was

103 purchased from Qiagen (Germantown, MD). High Capacity cDNA RT kit were purchased from

104 ThermoFisher Scientific (Waltham, MA). RT² SYBR Green ROX qPCR Mastermix and primers

105 for Gapdh were purchased from Qiagen (Valencia, CA).

106 2.2 Animals and treatment

107 This study was approved by Iowa State University Animal Care and Use Committee. Seven- to

108 eight-week-old male C57 BL/6J mice were housed at room temperature of 20 - 22 °C under a 12-

109 h light cycle, and a relative humidity of 35 – 50 %. Protein Rodent maintenance diet (Teklad

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110 HSD Inc, WI, US) and water were provided ad libitum. Prior to exposure on day 1, all mice were

111 acclimated to breathing air for 40 min on each of two days preceding day 1 when mice were first

112 exposed to H2S. Mice were exposed by whole body inhalation exposure either to normal

113 breathing air from a tank under pressure (Control) or to 650 - 750ppm H2S (H2S-treated) once or

114 for short-term daily exposures lasting for up to 6 days. The exposure paradigm is summarized in

115 Fig 1. On day 1, mice were exposed to H2S of 650 - 750 ppm for 40 min only. Those mice

116 terminated on day 1 were euthanized 2 h post H2S-eposure. The remaining groups of H2S-

117 exposed mice were exposed daily to 765 ppm H2S for 15 min. only. In this regard, mice

118 euthanized on day 1 received only 1 acute exposure, those euthanized on day 2 received 2 acute

119 exposures of H2S and those euthanized on day 4 received 4 acute exposures to H2S. Negative

120 controls were exposed to normal breathing air daily and euthanized on day 4. Mice were

121 euthanized by decapitation 2 h after the last exposure to H2S on days 1, 2 and 4 for proteomics

122 analysis and quantitative gene expression analysis by real-time PCR and on days 1, 3 and 7 for

123 histopathology evaluation. Following decapitation, brains were immediately removed from the

124 skull. The IC were microdissected on ice and immediately flash frozen using liquid nitrogen, and

125 stored at -80°C until further use. Animals were cared for in accordance with the Institutional

126 Animal Care and Use committee guidelines.

127 2.3 Behavioral assessment

128 The VersaMax open field test was used to assess motor deficits induced by H2S. We used this

129 test because previous studies in the lab had indicated it to be sensitive to acute H2S intoxication

130 in this mouse model (Anantharam et al., 2017). Spontaneous activity was measured using an

131 automated computer device (Model RXYZCM-16; Accuscan, Columbus, OH, USA). The

132 activity chamber’s dimensions are 40 x 40 x 30.5 cm, and it is made of Plexiglas with a Plexiglas

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133 lid. The lid has holes for ventilation. Data was analyzed using VersaMax Analyzer (Model CDA-

134 8; Accuscan). Mice were placed in the activity chamber 2 min prior to recording for 10 min, for

135 acclimation. Vertical activity, horizontal activity, and distance travelled were measured.

136 2.4 Histopathology

137 Mice were euthanized 24 h after the last H2S exposure. They were deeply anesthetized with a

138 cocktail of 100 mg/kg bw ketamine and 10 mg/kg bw xylazine. The thoracic cavity was opened

139 to expose the heart and fresh 4 % paraformaldehyde (PFA, pH 7.4) was injected through the left

140 ventricle. After perfusion, the calvarium was opened and brains were post-fixed in 4 % PFA for

141 24-48 h before removal from the skull. Brains were processed in paraffin, sectioned at 5 microns,

142 and stained with hematoxylin and eosin, and examined microscopically. Neurons were stained

143 with NeuN antibody (ab177487, Abcam, Cambridge, MA) using an indirect immunostaining

144 protocol. Diaminobenzidine was used as chromogen. Stained sections were examined using a

145 Nikon Eclipse Ci-L microscope equipped with a DS-Fi2 camera. For image analysis for the

146 quantification of neurons, NeuN-positive cells (sites of DAB chromogen deposition) were

147 enumerated in each of five 400X photomicrographs of the IC from mice exposed to H2S or

148 breathing air, and the mean number of NeuN-positive cells/mouse were compared between

149 groups.

150 2.5 Mass Spectrometry Analysis

151 Samples were processed according to a previously published method with some modifications

152 (Meade et al., 2015). Briefly, IC tissues were placed in 100µl of urea lysis buffer and

153 homogenized with a handheld pestle homogenizer. Protein samples were further reduced and

154 alkylated. Small aliquots of each sample were taken to measure protein concentration using the

155 Bradford assay from Bio-Rad (Hercules, CA). The remaining samples were diluted and trypsin-

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156 digested overnight, followed by desalting using a C18 peptide trap from Michrom (Auburn, CA).

157 The desalted samples were vacufuged prior to individual sample labeling using TMT-6plex

158 labels according to the manufacturers’ instructions (Thermo FisherScientific, Waltham, MA).

159 Labeled samples of each exposure group were combined with the control for sample comparison

160 (Table 1). Peptides were separated on a Waters BEH C18 capillary column prior to online

161 analysis using a 240-min linear increasing gradient of acetonitrile with 0.1% formic acid.

162 Following elution from the column, ions were generated using 2.6 kV on a taper tip in a New

163 Objective nanosource and entered into an LTQ-OrbitrapVelos mass spectrometer (Thermo

164 Fisher, San Jose, CA). A full scan was taken in the LTQ, followed by data-dependent MS/MS

165 analysis of the top 6peaks. MS/MS analysis included collision-induced dissociation (CID) in the

166 LTQ for structural information and higher-energy collisional dissociation (HCD) in the Orbitrap

167 for quantitation. MS/MS data were aligned and quantitated using MaxQuant1.5.4.1 (Cox and

168 Mann, 2008) Analytics Platform with PTXQC (Bielow et al., 2016) quality control data

169 management. Peptide alignment was executed with the mouse Uniprot protein database

170 UP000000589_10090.fasta; enzyme: trypsin; carboxymethyl (C), and oxidation (M), FDR <1%

171 based on peptide q-value under standard settings (see supplemental data). The secondary

172 computational analysis was executed using Perseus 1.5.5.3 (Tyanova et al., 2016) for statistical

173 rendering, and web-based software Morpheus for heatmap rendering. Prior to analysis of

174 experimental samples, small aliquots of individually labeled control samples were analyzed to

175 determine individual variations in controls. Due to low variability in the controls, a pooled

176 control setting was used. Sample analysis included modification of the MaxQuant protein output

177 list by normalization of individual TMT reporter ion intensities by division through the median

178 intensity followed by determination of fold TMT expression ratios by division of individual

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179 experimental TMT values (126,127,128,129,and 130) through the TMT value (131) of the

180 pooled control.

Total Mice/ TMT Exposure/ Group Treatment Euthanasia Animal ID Label Dose

(n=5) 40 min day 1 Inhalation

1,2,3,4,5, 131 15 min day 2 / GA Day 4 pooled 15 min day 3 air Control 15 min day 4

+ 2 h recovery

(n=5) 40 min day 1 Inhalation

GB 1 126 + 2 h recovery /

1 2 127 650 - 700 Day 1

exposure 3 128 ppm H2S

4 129

5 130

(n=5) 40 min day 1 Inhalation

1 126 15 min day 2 / GC 2 127 + 2 h recovery 650 – 700 2 Day 2 3 128 ppm H2S exposures 4 129

5 130

GD (n=4) 40 min day 1 Inhalation Day 4 4 1 126 15 min day 2 /

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exposures 2 127 15 min day 3 650 – 700

3 128 15 min day 4 ppm H2S

5 130 + 2 h recovery

181 Table 1. Animal exposure paradigm for mass spectrometry and distribution of TMT sample

182 labeling. GA= group A; GB= group B; GC=group C; GD= group D. (two column table)

183 2.6 Perseus Statistical Analysis

184 The previously modified Maxquant protein list was entered into the Perseus 1.5.5.3

185 software program followed by annotating Maxquant defined Swissprot protein accession

186 numbers with mouse gene identifiers. Statistical analysis included one-sample t-tests of fold

187 expression values by considering the deviation of samples from 1 fold expression (no change in

188 TMT reporter ion intensities vs. control intensity values) using p<0.05 as a criterion for

189 significance. Scatter Plots were established by plotting one-sample t-test difference fold protein

190 expression vs. –log one-sample t-test p value fold protein expression according to the associated

191 Perseus tool set.

192 2.7 Morpheus Heatmap Rendering

193 The Perseus processed protein list containing mouse gene identifiers, protein names and gene

194 ontology (GO) biological process, one-sample t-test, levels of significantly and non-significantly

195 modulated proteins was entered into the web-based Morpheus heatmap-rendering tool

196 (https://software.broadinstitute.org/morpheus/). A gradient coloring scheme was applied to

197 display upregulated (above 1.2 fold expression (in log2 display above 0.263), red color) vs.

198 downregulated (below 0.83 fold expression (in log2 display below -0.263) blue color) protein

199 nodes. White color nodes represent changes of non-modulated nodes to control levels (between

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200 0.83 and 1.2 fold expression). The heatmap was hierarchically clustered by Euclidean distance

201 using row average linkage and grouping of rows by GO biological process.

202 2.8 Validation of gene expression via quantitative real-time RT-PCR

203 After mice were exposed to H2S, tissues were dissected and immediately stored at -80 °C till

204 analysis. Total RNA was extracted from frozen tissues using the RNeasy® Plus Mini kit with

205 treatment of DNase I according to the manufacturer’s protocol. Validated primers for Gapdh

206 (Qiagen, #PPR57734E) were used as the housekeeping gene controls. The threshold cycle (Ct)

207 was calculated from the instrument software, and fold change in gene expression was calculated

208 using the Ct method as described earlier (Kim et al., 2016). The following primers were used

209 to check the quantitative transcriptional level of Prkab1, Vim, and Ahsa1; 5’-

210 TCCGATGTGTCTGAGCTGTC-3’ and 5’-CCCGTGTCCTTGTTCAAGAT-3’ for Prkab1

211 (Bandow et al., 2015), 5’-TCCACACGCACCTACAGTCT-3’ and 5’-

212 CCGAGGACCGGGTCACATA-3’ for Vim (Ulmasov et al., 2013), 5’-

213 CAGAGGGGCACTTTGCCACCA-3’ and 5’-CACGGCCTTCCATGCACAGCT-3' for Ahsa1.

214 2.9 Statistical analysis

215 Data were analyzed using Prism 4.0 (GraphPad Software, San Diego, CA). Non-paired

216 Student’s t-test was used when two groups were being compared. Differences were considered

217 statistically significant for p-values <0.05. Data are represented as the mean ± S.E.M. of at least

218 two separate experiments performed at least in triplicate.

219 3. Results

220 3.1 Acute exposure to H2S induces motor behavioral deficits and seizures in C57 black mice.

221 Locomotor activities of mice were assessed on days 2, 4, and 6. Results indicated that the

222 horizontal and vertical activities were decreased by more than 50% and were statistically

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223 different in mice exposed to acute H2S compared to the breathing air group (Fig. 2 A and B).

224 Total distance traveled was also decreased by more than 50% and was also statistically different

225 in H2S-exposed mice compared to controls (Fig. 2 C). More importantly, mice exposed to H2S

226 exhibited severe seizure activity. On day 1, on average, seizure activity was observed starting at

227 15 min of H2S exposure. More than 50% of mice were shown to have seizure activity at 40 min

228 of exposure to H2S. Mice exposed to H2S more than once exhibited seizure activity after only 5

229 min of H2S exposure. In these groups of mice, at 10 min of exposure to H2S, 40% of mice

230 exhibited seizure activity on day 6. Collectively, these results indicate increased susceptibility of

231 mice to H2S after each successive H2S exposures, suggesting that the toxic effects of H2S

232 exposure are cumulative (Fig. 2D).

233

234 Figure 1. Acute exposure paradigm of hydrogen sulfide on C57 black mice.

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235 Mice were exposed to 765 ppm of hydrogen sulfide in a chamber for 40 min either once only

236 (day 1) and for 15 min on the following days up to day 7. Mice were sacrificed 2 h post-H2S

237 exposure on specified days of the study. Negative control mice were exposed to breathing air

238 from a cylinder daily up to day 7. Separate groups of mice were sacrificed on days 1, 3, and 7 for

239 immunohistochemistry. Groups of mice for proteomics studies were sacrificed on days 1, 2 and 4.

240 (One column figure)

241

242 Figure 2.Acute exposure to hydrogen sulfide induced motor behavioral deficits.

243 C57 black mice were exposed to H2S as shown in Fig. 1. Locomotor activity was measured using

244 an automated VersaMax locomotor activity monitor during 10 min time test on days 2, 4, and 6.

245 Horizontal activity (A), vertical activity (B), and total distance traveled (C) were analyzed

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246 between groups. Seizure activity was analyzed on a time scale (D). Asterisks (*, p < 0.05; **, p <

247 0.01) indicate statistically significant differences between H2S and breathing air negative control

248 groups. (One and half column figure)

249

250 3.2 Acute hydrogen sulfide exposure induces brain damage

251 Motor behavioral deficits induced by exposure to H2S may be a sign of injury to the central

252 nervous system (CNS). Therefore, histopathology was performed to identify the effects of H2S

253 exposure in the IC. Mice were exposed to H2S as designated in Fig. 1 and sacrificed at multiple

254 time points (day1, 3, or 7) for this portion of study. Microscopic examination of PFA-perfused

255 brains revealed H2S-induced neurodegeneration and loss of neurons in the IC (Figure 3A). By

256 day 7, mice exposed to H2S exhibited severe damage to the IC, with necrosis, vacuolar change,

257 and infiltration by neuroglial cells. In order to further characterize loss of neurons, IC tissues

258 were analyzed by immunohistochemical staining for the neuron specific marker, NeuN. On day 7,

259 there was a marked loss of neurons in the IC of H2S-exposed mice, compared with the breathing

260 air group. Enumeration of neurons in the IC revealed approximately 70 % loss of IC neurons by

261 day 7 of H2S exposure (Fig. 3 B and C) with infiltration by glial cells. In H2S exposed mice,

262 neurons in the midbrain adjacent to the IC appeared unaffected morphologically and glial cell

263 numbers and activation state were not altered. These results demonstrate selective loss of

264 neurons of the IC.

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265

266 Figure 3. Neurodegeneration and necrosis in the IC of mice exposed to 650-750 ppm H2S in

267 acute short-term repeated inhalation exposures over 7 days. Note the loss of neurons and

268 development of clear vacuoles in the neuropil (arrow) on day 7 in mice exposed to H2S and the

269 minimal morphologic effects to earlier H2S exposures or breathing air (A). Inhalation of H2S

270 resulted in marked and selective loss of neurons in the IC (1000X magnification images, B), with

271 retention of neurons in the regions surrounding the IC (200X magnification image). Computer

272 aided image analysis of NeuN immunostained sections reveals marked loss of neurons in the IC

273 of mice exposed to H2S (p=0.001, Students t test). Representative photomicrographs of mice

274 exposed to breathing air or H2S, hematoxylin and eosin (A) and NeuN immunohistochemistry

275 (B), 400X magnification (A) and 1000X and 200X magnification (B). Neurons in the IC region

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276 of the H2S exposed mice on day 7 were enumerated and compared to breathing air control (C).

277 (Two column figure)

278 3.3 Changes in the Broad Spectrum Proteome following acute H2S Exposure

279 Proteomic profiles of the IC were determined using TMT peptide labeling coupled with

280 LC/MS/MS analysis by comparison of the breathing air negative control group and the H2S

281 exposed group is described in the methods section. Mass spec was able to identify 598, 562, and

282 546 altered proteins for days1, 2, and 4 of H2S exposure, respectively. Subsequent analyses

283 showed alterations of protein expressions (Fig. 4, heatmap) with 36, 12 and 13 proteins being

284 significantly downregulated (below 0.83 fold) and 8, 6, and 12 proteins being significantly

285 upregulated (above 1.2 fold) compared to the control for days 1, 2 and 4 of H2S exposure,

286 respectively. A single (day 1) H2S exposure, in which mice were euthanized only 2 h post a

287 single exposure, demonstrated the highest range of fold protein expression changes compared to

288 the control with the majority of proteins being in the downregulation cluster. This was followed

289 by day 2 exposures with equal distribution of upregulated and downregulated proteins, while day

290 4 manifested the lowest range of fold changes with the least changes in the proteomic profile

291 (Fig. 5 A, B, C, D, scatter plots).

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292

293 Figure 4.Heatmap of IC changes in proteomic profile following H2S exposure

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294 Morpheus rendered Heatmap of determined changes in protein expression on days 1, 2 and 4.

295 H2S exposure using hierarchical clustering by Euclidean distance, row average linkage and

296 grouping of rows by gene ontology for biological process (GOBP). Heatmap displays fold

297 expression values of five (day 1 and day 2) or four (day 4) IC tissue samples, and average fold

298 expression values, mouse gene identifiers (gene names), protein names, and GOBP. “+”

299 indicates t-test (of fold expression values) significantly modulated proteins; grey not identified,

300 white non regulated, red upregulated (above 1.2 fold expression vs. control), blue downregulated

301 (below 0.83 fold expression vs. control) converted into a log2 data display. (Two column figure)

Gene Ontology Term Day1 Day2 Day4

actin cytoskeleton organization 3 4 2

adhesion 1 0 1

anion transport 1 0 0

axon guidance 3 0 1

blood 1 2 1

cell cycle 1 0 1

cellular transport 1 0 1

DNA metabolism 1 2 0

energy metabolism 6 3 5

glucose metabolism 3 3 1

immune response 1 1 1

microtubule organization 1 1 2

mitochondrial transport 1 0 0

oxidoreductase 1 0 0

protein degradation 1 0 0

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protein folding 3 1 0

RNA metabolism 7 2 3

signaling 6 2 2

vesicle transport 3 3 2

fatty acid steroid metabolism 0 1 1

mitochondrial proteolipid 0 1 1

synaptic transmission 0 1 1

tight junction component 0 1 0

carbohydrate transport 0 0 1

lipid metabolism 0 0 1

phosphatidylethanolamine biosynthesis 0 0 1

translation 0 0 1

lysosomal proton pump 0 1 1

302

303 Table 2. Functional annotation of significantly modulated protein expression in the IC following

304 H2S exposure on days 1, 2, and 4. Table displays total number of proteins associated with GO-

305 term definition. A gradient scheme was applied to better indicate number distribution with lower

306 numbers in light orange and higher numbers in dark orange. Note that day 1 represents mice

307 exposed once and euthanized 2 h post-exposure. (One and half column table)

308 We further analyzed H2S-dependent alterations in the proteome using Perseus annotation for

309 gene ontology (GO) biological pathways that were summarized into general categories when

310 applicable by integrating gene cards information (www.genecards.org). We found that several

311 gene ontology functions were affected after H2S exposure (Table 2). Acute exposure to H2S on

312 day 1 resulted in alteration of RNA, energy and glucose metabolism, and changes in signaling

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313 pathways, axon guidance, actin cytoskeleton reorganization, and vesicle transport. On day 2

314 changes in gene ontology included glucose and energy metabolism, actin cytoskeleton

315 organization and vesicle transport. Changes in energy and RNA metabolism were most abundant

316 on day 4. Interestingly, there was an upregulation of cytoplasmic interacting protein Cyfip2 and

317 nuclear hormone receptor and transcriptional repressor family Nr1d2 that were observed at all

318 three time points. There was also a downregulation of anti-apoptotic and immunogenic protein

319 Bcl2 associated athanogene Bag5 on day 1, suggesting potential induction of apoptosis. This is

320 further supported by an observed downregulation of core histones H3f3a, and Hist1h3a that

321 might result in a DNA damage response. Evidence of potential immunogenic changes was given

322 by an upregulation in complement binding protein C1qbp on day 3 and a downregulation in anti-

323 inflammatory protein macrophage migration inhibition factor Mif on day 1. These latter changes

324 suggest inflammation is involved in H2S-induced neurotoxicity.

325

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326 Figure 5.Overall distribution of IC changes in proteomic profile following H2S exposure

327 A)Scatter Plot to display overall distribution of one-sample t-test differences in protein

328 expression profiles vs. –Log one-sample t-test p values following day 1 (red), day 2 (blue), and

329 day 4 (green) H2S exposure. Scatter Plots of one-sample t-test fold protein expression differences

330 vs. –Log one-sample t-test p values from B) day 1, C) day 2, and D) day 4 of H2S exposure.

331 One-sample t-test of significantly modulated proteins in the H2S exposure group vs. the control

332 according to fold expression values are displayed with official mouse gene symbols in red. (Two

333 column figure)

334

335 3.4 Validation of proteomic changes of genes after H2S exposure

336 To confirm the observed changes in the IC proteomic profile following acute exposure to H2S,

337 several genes were analyzed by quantitative RT-PCR. Mice were sacrificed 2 h after each

338 designated time point to measure cellular response right after H2S exposure. Protein kinase

339 AMP-activated non-catalytic subunit beta 1 (Prkab1, also called Ampk) is known to correlate

340 with calcium fluctuations as a measure of cellular calcium response (Yong et al., 2010). Prkab1

341 mRNA expression was analyzed as a means to measure the calcium-dependent cellular response

342 following H2S exposure. Prkab1mRNA expression was upregulated following H2S exposure on

343 day 1, 2 and 4 which is in line with the mass spec observed upregulation of protein Kinase

344 cAMP-activated catalytic subunit alpha (Prkca) and calcium activated protein kinase C beta

345 (Prkacb) on exposure day 1. To confirm potential H2S exposure dependent pro-inflammatory and

346 ischemic effects, mRNA expression of Vimentin (Vim) was measured. Vim mRNA expression

347 demonstrated a steady increase throughout all days of H2S exposure (Fig. 6 B). This is further

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348 supported by the increased protein expression of Vim on exposure day 1, while mass spec lacked

349 detection of Vim in the other samples.

350 Due to a mass-spec observed downregulation in the activator of 90 kDa heat shock protein and

351 chaperone ATPase homlog1 (Ahsa1) on day 1 and 2, mRNA expression of Ahsa1 was examined

352 by RT-PCR (Fig. 6 C). Similar to the mass spec observed protein expression, mRNA expression

353 of Ahsa1 demonstrated a significant 30-40 % downregulation on days 1 and 2 of H2S exposure,

354 compared to the breathing air control group.

355

356 Figure 6.Validation of expression changes of three genes at the mRNA level in IC after H2S

357 exposure. The transcriptional level of three genes (A; Prkab1, B; Vim, C; Ahsa1) was measured

358 by quantitative PCR. Samples were normalized with the reference gene Gapdh. Data are

359 represented as the mean ± S.E.M. Note that only a single acute exposure to H2S was needed to

360 cause upregulation of gene expression of PkAb1 and Vim or downregulation of Ahsa1 gene

361 expression. (Two column figure)

362

363 4. Discussion

364 Acute H2S exposure often leads to neurological sequelae among human survivors, suggesting a

365 trigger of neurodegeneration cascade following acute H2S exposure (Matsuo et al., 1979; Tvedt

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366 et al., 1991a; Tvedt et al., 1991b; Kilburn, 1993; Snyder et al., 1995; Woodall et al., 2005).

367 However, the exact cellular and molecular mechanisms underlying delayed neurological

368 disorders after acute H2S exposure are poorly understood. Few studies have been done to

369 characterize mechanisms of H2S exposure-induced neurotoxicity in survivors of acute H2S

370 poisoning. We recently developed an inhalational mouse model which recapitulates H2S-induced

371 neurotoxicity observed in humans (Anantharam et al., 2017). In this model, mice exposed to H2S

372 showed clinical and pathological characteristics as shown in human patients. In case studies of

373 human H2S inhalation exposure, H2S concentrations > 500 ppm were reported to rapidly induce

374 severe neurotoxicity and temporary paralysis of breathing, leading to immediate collapse

375 (Guidotti, 1996; Chou et al., 2016). Survivors of acute high H2S exposures (> 500 ppm)

376 developed chronic neurological problems such as loss of memory, loss of control of facial

377 muscles, persistent headaches, and neurodegeneration. These patients also exhibited motor

378 behavioral deficits such as spastic gait (Wasch et al., 1989; Tvedt et al., 1991a; Tvedt et al.,

379 1991b; Callender et al., 1993). Brain scan images of patients accidently exposed to H2S

380 suggested a dysregulation of basal ganglia region that governs motor behavior (Schneider et al.,

381 1998; Nam et al., 2004). This is consitent with the observed motor deficits in our RotaRod

382 studies that are in agreement with clinical phenotypes of H2S exposed human patients. According

383 to Wasch et al., the brainstem was demonstrated to accumulate the highest concentration of H2S

384 compared to other brain regions in the rat (Wasch et al., 1989). We have previously identified

385 and herein confirmed that the IC, a brainstem region, is highly sensitive to H2S-induced necrosis.

386 The relative sensitivity of the IC to H2S-induced injury reflects the unique elements and cell

387 populations in this region of the brain. We observed histologic features of neurodegeneration,

388 neuronal death, and reactive gliosis is in the IC starting on day 3 with massive cell necrosis and

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389 glial scarring in the IC on day 7. Immunohistochemistry with neuron specific antibody (NeuN)

390 revealed severe neuronal loss in the IC with H2S exposure. Loss of neuronal cells was more than

391 70 % compared to breathing air control group in IC. In contrast, brain stem regions adjacent to

392 the IC were shown to be relatively unaffected, indicating unique susceptibility of the IC region to

393 H2S toxicity. The function of the IC is to integrate auditory and other sensory signals and is

394 reportedly a region with high metabolic rate requirements (Ridgway et al., 2006; Houser et al.,

395 2010) while it is also the most highly vascularized brain region (Gonzalez-Lima et al., 1997).

396 H2S is a systemic metabolic toxicant that reportedly interferes with ATP synthesis. It is

397 reasonable to infer that brain regions with high blood supply and metabolic rates such as the IC

398 are more vulnerable to H2S-induced toxicity.

399 One of the hypotheses underlying H2S-induced neurotoxicity is that H2S causes hypoxia-

400 dependent neurodegeneration following H2S induced hypotension and pulmonary edema-

401 dependent oxygen deprivation in inhalation victims of H2S poisoning (Nicholls and Kim, 1982;

402 Dorman et al., 2002; Miyazato et al., 2013; Rumbeiha et al., 2016). However, the proximate

403 molecular pathways by which these events lead to neurodegeneration are unknown. In this study,

404 we used proteomic analysis to define cellular and molecular mechanisms of H2S induced

405 neurodegeneration during early stages of injury. We demonstrated that H2S exposure induced

406 significant alteration of protein expression in the IC in various biological pathways, including

407 cellular morphology, energy metabolism, and calcium signaling. It was most interesting that a

408 single H2S exposure exerted the most significant changes, suggesting a single exposure, as

409 commonly occurs during single accidental H2S exposures, is sufficient to trigger such proteomic

410 changes.

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411 Energy failure is another hypothesis of H2S-induced neurotoxicity because it exerts its cellular

412 toxicity by inhibition of cytochrome C oxidase which plays a crucial role in mitochondrial ATP

413 synthesis (Nicholls and Kim, 1982; Dorman et al., 2002). In this study, expression levels of

414 cytochrome C oxidase-related proteins were not changed, while cytochrome C oxidase subunit 6

415 C (Cox6c) and mitochondrial cytochrome C oxidase subunit 2 (mt-Co2) were decreased on day 4

416 and 1, respectively, supporting potential impairment of energy production following H2S

417 exposure. Several other proteins related to energy production were also altered. For instance,

418 ATP synthase subunits Atp5j2 and ATP5l together with cytochrome C oxidases 6C were

419 downregulated on days 2, and 4, respectively. In addition, citric acid cycle proteins malic

420 enzyme (Me1) and phosphoglycerate mutase (Pgam2) were downregulated on day 1. Further

421 studies are warranted to check whether Coxy6c and mt-Co2 are the bona fide targets of H2S

422 toxicity. Collectively, these results suggest hypoxia, resulting in low oxygen tissue delivery, and

423 impaired ATP synthesis arising from dysregulation of cytochrome C related proteins, likely work

424 in concert leading to neuronal death in the IC.

425 The IC undergoes severe degenerative changes during H2S exposure including widespread

426 neuronal loss, which is regionally selective. Long-term exposure to H2S was demonstrated to

427 result in neuronal structural damage by demyelination (Solnyshkova, 2003). Interestingly,

428 claudin-11 (Cldn11), a major tight junction component in neuronal myelin structures of the

429 central nervous system (CNS) was downregulated following day 1 and 2 of H2S exposure

430 supporting potential H2S induced neurodegeneration (Gow et al., 1999; Tiwari-Woodruff et al.,

431 2001). Besides, mass spectrometry detected alterations in proteins related to cellular

432 morphological changes included regulators of actin cytoskeleton and microtubule organization.

433 Dynamine-3 (Dnm3), a GTP binding protein associated with the microtubule system was

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434 downregulated on day 1. Dynein Axonemal Heavy Chain 6 (Dnah6), a component of

435 microtubule-associated motor system, was upregulated on day 4. Moreover, a couple of adhesion

436 proteins were altered, e.g., vimentin (Vim) was upregulated on day 1 while prelaminin-A/C

437 (Lmna) was upregulated on day 4 of H2S exposure. Dihydropyrimidinase-related protein Dpysl4,

438 and neurofilament light polypeptide Nefl were downregulated on day 1 together with a

439 downregulation of myelin proteolipid protein Plp1 throughout the study period. With regards to

440 other cytoskeletal proteins, Tuba1b and Tubb6 were downregulated on day 4. These changes

441 may impact cell integrity and function in the IC, leading to neuronal death.

442 H2S exposure has previously been shown to induce dysregulation of calcium concentrations

443 (Garcia-Bereguiain et al., 2008). It was further reported by others that exposure to H2S may

444 affect regulation of calcium (Nagai et al., 2004; Garcia-Bereguiain et al., 2008; Yong et al.,

445 2010). In this study, the calcium binding protein neurogranin (Nrgn) demonstrated a non-

446 significant upregulation on day 1. However, we found a significant upregulation in cAMP-

447 dependent protein kinase that matched the expression pattern of Prka1 mRNA supporting

448 potential activation of calcium-dependent signaling. These results agree with previous reports

449 that demonstrated H2S-dependent calcium and cyclic-AMP signaling through Prka (Kimura,

450 2000; Yong et al., 2010). Collectively, these results suggest calcium dysregulation as a potential

451 mechanism of H2S-induced neurotoxicity.

452 Changes in immunogenic biomarkers represent one of earliest responses to cytotoxicity.

453 Evidence of potential H2S-dependent immunogenic changes was given by a downregulation in

454 anti-inflammatory protein macrophage migration inhibition factor Mif on day 1 and an

455 upregulation in the reactive oxygen species (ROS) response and complement binding protein

456 C1qbp on day 4. Immunogenic changes in MIF have been previously observed (Roger et al.,

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457 2003). ROS response and complement binding protein C1qbp has been previously reported

458 (McGee and Baines, 2011). In addition, there was an upregulation of cytoplasmic interacting

459 protein (Cyfip2) and nuclear hormone receptor and transcriptional repressor family (Nr1d2) that

460 were observed throughout the exposure period. Cyfip2 was previously shown by others to have a

461 p53-response element in its promoter region and to be one of the direct targets of p53 (Jackson et

462 al., 2007). Besides, the anti-apoptotic and immunogenic protein Bcl2 associated athanogene

463 (Bag5) was downregulated on day 1. Endoplasmic reticulum stress-induced downregulation of

464 Bag5 has previously reported (Bruchmann et al., 2013; Gupta et al., 2016). Further evidence of

465 potential cell stress-dependent impairment in protein folding was shown by an RT-PCR and

466 mass spec detected downregulation in Ahsa1 that plays a role as chaperone and activating heat

467 shock protein 90 (Okayama et al., 2014; Tripathi et al., 2014). These results indicate that

468 inflammation may play an important role in H2S-induced neurotoxicity.

469 H2S-induced neurotoxicity has been suggested to resemble the injury caused by ischemic

470 hypoxic conditions (Doujaiji and Al-Tawfiq, 2010; Rumbeiha et al., 2016). Previous studies

471 have suggested neurofilament Nefl, and collapsin Response Mediator Protein 3 (Dpysl4) as

472 biomarkers of hypoxia and cerebral ischemia (Hou et al., 2006; Lian et al., 2015). Dpysl4 is

473 cleaved by activated calpain reaction in response to cerebral ischemia (Hou et al., 2006). Both

474 Nefl and Dpysl4 were downregulated during H2S exposure. In addition, mass spec detected

475 downregulation of phosphoglycerate mutase 2 (Pgam2), an ischemia biomarker, on day 1. This

476 biomarker is suggested to correlate with the ischemic conditions (Li et al., 2012). Vimentin (Vim)

477 has been shown as another biomarker for ischemia (Li et al., 2008). Mass spec also detected a

478 steady increase in protein and mRNA expression of the inflammatory and ischemia biomarker

479 Vim for the entire duration of the study. These data support ongoing progression of H2S-

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480 dependent ischemia-like conditions during acute H2S exposures, further supporting the role of

481 hypoxia as a mechanism of H2S-induced neurotoxicity.

482

483 5. Conclusion

484 In this study we examined the early effects of acute H2S exposure on brains of mice following

485 either a single H2S exposure or up to 4 short-term acute exposures. Taken together, acute

486 exposure to H2S induced neurotoxicity, which manifested as progressive behavioral deficits. The

487 IC was the most sensitive brain region, confirming our previous observations. Results point to

488 demonstrated modulation of several proteome-based biological pathways in the IC. Specifically,

489 results of proteomic and gene expression studies suggest calcium dysregulation, immune

490 mediated inflammatory response, pro-apoptosis mechanisms, and ischemia-like cytotoxicity to

491 be involved in H2S-induced neurotoxicity. This proteomic data provided important clues on

492 mechanisms of H2S-induced neurological toxicity. Further research is recommended to better

493 understand the singular or collective role of these potential mechanisms in H2S-induced mortality,

494 neurotoxicity, and evolution of neurological sequelae.

495

496

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497 Figure 1. Exposure paradigm of hydrogen sulfide on C57 black mice.

498 Mice were exposed to 765 ppm of H2S in a chamber for 40 min either once only (day 1) and for

499 15 min on the following days up to day 7. Mice were sacrificed 2 h post-H2S exposure for each

500 designated time point. Negative control mice were exposed to breathing air from a cylinder daily

501 up to day 7. Separate groups of mice were sacrificed on days 1, 3, and 7 for

502 immunohistochemistry. Groups of mice for proteomics studies were sacrificed on days 1, 2 and 4.

503 504 Figure 2. Exposure to hydrogen sulfide induced motor behavioral deficits.

505 C57 black mice were exposed to H2S as shown in Fig. 1. Locomotor activity was measured using

506 an automated VersaMax locomotor activity monitor during 10 min time test on days 2, 4, and 6.

507 Horizontal activity (A), vertical activity (B), and total distance traveled (C) were analyzed

508 between groups. Seizure activity was analyzed on a time scale (D). Asterisks (*, p < 0.05; **, p <

509 0.01) indicate significant differences between H2S and breathing air negative control groups.

510 511 Figure 3. Neurodegeneration and necrosis in the inferior colliculus of mice exposed to 650-750

512 ppm H2S in short-term repeated acute inhalation exposures over 7 days. Note the loss of neurons

513 and development of clear vacuoles in the neuropil (arrow) at day 7 in mice exposed to H2S and

514 the minimal morphologic effects to earlier H2S exposures or breathing air (A). Inhalation of H2S

515 resulted in marked and selective loss of neurons in the IC (1000X magnification images, B), with

516 retention of neurons in the regions surrounding the IC (200X magnification image). Computer

517 aided image analysis of NeuN immunostained sections reveals marked loss of neurons in the IC

518 of mice exposed to H2S (p=0.001, Students t test). Representative photomicrographs of mice

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519 exposed to breathing air or H2S, hematoxylin and eosin (A) and NeuN immunohistochemistry

520 (B), 400X magnification (A) and 1000X and 200X magnification (B). Neurons in the IC region

521 of the H2S exposed mice on day 7 were enumerated and compared to breathing air control (C).

522

523 Figure 4. Heatmap of IC changes in proteomic profile following H2S exposure

524 Morpheus rendered Heatmap of determined changes in protein expression following 1, 2 and 4

525 day H2S exposure using hierarchical clustering by Euclidean distance, row average linkage and

526 grouping of rows by gene ontology for biological process (GOBP). Heatmap displays fold

527 expression values of five (day 1 and day 2) or four (day 4) analyzed IC tissue samples, and

528 average fold expression values, mouse gene identifiers (gene names), protein names, and GOBP.

529 “+” indicates t-test (of fold expression values) significantly modulated proteins; white non

530 regulated, red upregulated (above 1.2 fold expression vs. control), blue downregulated (below

531 0.83 fold expression vs. control) converted into a log2 data display.

532

533 Figure 5. Overall distribution of IC changes in proteomic profile following H2S exposure

534 A) Scatter Plot to display overall distribution of one-sample t-test differences in protein

535 expression profiles vs. –Log one-sample t-test p values following day 1 (red), day 2 (blue), and

536 day 4 (green) H2S exposure. Scatter Plots of one-sample t-test fold protein expression differences

537 vs. –Log one-sample t-test p values from B) day 1, C) day 2, and D) day 4 H2S exposure. One-

538 sample t-test of significantly modulated proteins in the H2S exposure group vs. the control

539 according to fold expression values are displayed with official mouse gene symbols in red.

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540 Figure 6. Validation of expression change of three genes at the mRNA level in IC after H2S

541 exposure. The transcriptional level of three genes (A; Prkab1, B; Vim, C; Ahsa1) was measured

542 by quantitative PCR. Samples were normalized with the reference gene Gapdh. Data are

543 represented as the mean ± S.E.M. Note that only a single acute exposure to H2S caused

544 upregulation of gene expression of PkAb1 and Vim or downregulation of Ahsa1 gene expression.

545 546 547 Acknowledgment: The authors do not have any conflict of interest. This work was partially

548 supported by the Iowa State University College of Veterinary Medicine Seed grant, Startup funds

549 and incentive account funds for Rumbeiha.

550 551 552

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