Effect of KNL1 on the Proliferation and Apoptosis of Colorectal Cancer Cells

bioRxiv preprint doi: https://doi.org/10.1101/499889; this version posted December 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Effect of KNL1 on the proliferation and apoptosis of colorectal cancer cells

2 Tianliang Bai1, Yabin Liu1, Binghui Li1*

3 1Department of General Surgery, Fourth Affiliated Hospital, Hebei Medical University,

4 Shijiazhuang, Hebei 050011, P.R. China.

5 *Corresponding author: Professor Binghui Li, Department of General Surgery,

6 Fourth Affiliated Hospital, Hebei Medical University, 12 Jiankang Road,

7 Shijiazhuang, Hebei 050011, P.R. China, E-mail: [email protected]

8 Tel: +86-031186095347

9 Fax: +86-031186265557

10 Key words: Kinetochore scaffold 1, colorectal cancer, cell proliferation, apoptosis

30 Tianliang Bai et al: Effect of KNL1 on the proliferation and apoptosis bioRxiv preprint doi: https://doi.org/10.1101/499889; this version posted December 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

31 Abstract.

32 Background: To identify the expression of KNL1 in colorectal tumor tissues and to

33 clarify the function of this gene on the proliferation capability of colorectal cancer

34 cells.

35 Methods: Thirty-six pairs of colorectal tumor and normal tissue were collected and

36 subjected to quantitative PCR analysis. KNL1 was under-expressed using lentiviral

37 transfection, and the function of KNL1 was evaluated by proliferation assay, colony

38 formation and apoptosis assay.

39 Results: KNL1 was highly expressed in colorectal tumor tissues. KNL1

40 downregulation inhibited colorectal cancer cell proliferation and promoted

41 apoptosis.

42 Conclusions: KNL1 plays an effective role in decreasing the apoptosis and promoting

43 the proliferation of colorectal cancer cells.

45 Introduction

46 Colorectal carcinoma (CRC) has been reported to be the second deadliest cancer 1-4.

47 Although the overall morbidity and mortality of CRC has decreased in recent decades,

48 there has been a considerable increase in patients <50 years of age 4-6. Additionally,

49 the 5-year survival of CRC is only 14% as the tumor is often not completely

50 resectable 5, 6. Despite advances in therapeutic strategies and diagnostic approaches,

51 the prognoses for patients with CRC typically remain poor 7. Therefore, there is an

52 urgent need to develop new CRC treatment approaches. We previously reported the

53 effects of D40 cloning on chromosome 15 8, and D40 was characterized to be a

54 cancer-related gene that is primarily expressed in the testis under normal conditions,

55 but is widely expressed in primary tumors of various origins as well as assorted

56 cancer cell lines 9, 10. A previous study has reported the mutual translocation of

57 AF15q14 with the MLL (Mixed lineage leukemia) gene in acute myeloid leukemia 11.

58 AF15q14, a human gene on chromosome 15, was also identical to the D40 gene 8.

59 Furthermore, D40 expression has been reported to be associated with the pathological

60 features of primary lung tumors, such as degree of differentiation, and patients’ Tianliang Bai et al: Effect of KNL1 on the proliferation and apoptosis bioRxiv preprint doi: https://doi.org/10.1101/499889; this version posted December 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

61 smoking history 10. In a study of the testes, D40 was found to be expressed in

62 pre-acrosomes and spermatocytes 12. A follow up study revealed that blinkin, a type of

63 kinetochore protein in the mitotic machinery was identical to KNL1. In addition to

64 binding of KNL1/blinkin with Ndc80 and Mis12 complexes, of which the KMN

65 network is comprised, binding is also observed between KNL1/blinkin and other

66 proteins, including tubulin, spindle assembly checkpoint (SAC) proteins BubR1 and

67 Bub1, and Protein Phosphatase 1 13-17. Such binding suggests that KNL1/blinkin has

68 crucial impacts on kinetochore formation in terms of the connection between spindles

69 and chromosomes, as well as the regulation of SAC 13-18.

70 Primary microcephaly (MCPH) is a rare congenital neurodevelopment disorder

71 that is known to have autosomal recessive features 19, 20. Due to different levels of

72 mental retardation, the head circumference of MCPH patients will be reduced and

73 their brains are smaller. However, additional somatic or neurological disorders are not

74 generally observed in patients with MCPH. MCPH is genetically heterogeneous and is

75 controlled by several chromosomal loci are responsible 19. In previous studies, an

76 MCPH locus has been reported on chromosome 15 20, and the gene responsible has

77 been identified as CASC5, which is also identical to KNL1 21. Considering that brain

78 development requires the rapid division of cells, this suggests that KNL1 is significant

79 for in vivo cell division.

80 In the present study, we analyzed KNL1 expression in various CRC and matched

81 adjacent noncancerous tissues, as well as in CRC cell lines. We also studied how

82 KNL1 knockdown impacts CRC cells in growing under an in-vitro in vitro by

83 transfecting the RKO and HT-29 CRC cell lines with shKNL1 or shCtrl.

84 As described previously 8, 10, 11, 13, 16, KNL1 has a number of alternative names,

85 including AF15q14, KIAA1570, CASC5, blinkin and D40. The nomenclature KNL1

86 was used in the present study.

88 Materials and methods

89 Patients and samples.

90 We collected human CRC and matching adjacent noncancerous tissue samples from

91 36 patients at the Department of General Surgery of the Fourth Hospital of Hebei

92 Medical University (Shijiazhuang, Hebei, China). All patients consented to inclusion

93 in the present study and all experiments were approved by the Ethics Committee of

94 the Fourth Hospital of Hebei Medical University.

96 Cell lines and cell culture

97 We purchased the human CRC cell lines (SW480, HT-29, HCT-116 and RKO) and the

98 normal colonic epithelial cell line NCM460 from the American Type Culture

99 Collection (ATCC; Manassas, VA, USA). Cells were maintained in RPMI-1640

100 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10%

101 fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere

102 containing 5% CO2 at 37°C.

103

104 RNA extraction and reverse transcription-quantitative polymerase chain

105 reaction (RT-qPCR).

106 We used TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) to extract whole

107 RNA from the sampled cells and tissues. RT was performed to generate cDNA using

108 M-MLV Reverse Transcriptase (Promega Corp., Madison, WI, USA) according to the

109 manufacturer’s protocol. A total of 1 µl cDNA was used as the template for qPCR.

110 GAPDH was used as an internal control. Primer sequences were as follows: KNL1,

111 forward 5'- ACA TTG GAA AAA GCG CAA GTTG-3' and reverse 5'- TTG CAC

112 TGG GCA ATA ATT GGC-3'; GAPDH, forward 5'-TGA CTT CAA CAG CGA CAC

113 CCA-3' and reverse 5'-CAC CCT GTT GCT GTA GCC AAA-3'. Thermocycling

114 comprised initial denaturation at 95˚C for 30 sec followed by 45 cycles of

115 denaturation at 95˚C for 5 sec and extension at 60˚C for 30 sec. The PCR products for

116 GAPDH and KNL1 were 121 and 213 bp in length, respectively. Each experiment

117 was performed in triplicate and relative quantitative gene expression was calculated as

118 previously described 22.

119 Tianliang Bai et al: Effect of KNL1 on the proliferation and apoptosis bioRxiv preprint doi: https://doi.org/10.1101/499889; this version posted December 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

120 Western blotting

121 Cells and tissues were lysed using RIPA buffer (Thermo Fisher Scientific, Inc.).

122 Lysates were electrophoresed by 10% SDS-PAGE (Thermo Fisher Scientific, Inc.)

123 and transferred onto PVDF membranes (Thermo Fisher Scientific, Inc.). After

124 blocking for 1 h in 5% skimmed milk, membranes were incubated overnight at 4°C

125 with rabbit antibodies against KNL1 (dilution, 1:500; Biorbyt Ltd., Cambridge, UK)

126 and GAPDH (dilution, 1:800; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

127 TBST was used to wash all the membranes 4 times. Membranes were subsequently

128 incubated at room temperature for 1 h with an HRP-conjugated secondary antibody

129 (dilution, 1:1,0000; catalog no., 323-065-021; Jackson ImmunoResearch, Inc., West

130 Grove, PA, USA). Protein bands were visualized using a chemiluminescence system

131 (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. The band

132 intensities were quantified using Image-Quant Software v3.0 (LI-COR Biosciences).

133 This experiment was repeated in triplicate.

134

135 Construction of recombinant lentiviral vector and cell transduction.

136 KNL1-shRNA and control-shRNA were designed by GeneChem Co., Ltd. (Shanghai,

137 China) from the unabridged sequence of D40/KNL1, which is targeted at the human

138 KNL1 gene (Genbank no. NM_144508). The targeting sequence of KNL1 was

139 5'-CAG AGT TGT ATG GTG GAAA-3'. To test how efficient KNL1 knockdown was,

140 stem-loop oligonucleotides were synthesized and inserted into a lentivirus-based

141 pGV115-GFP vector (GeneChem Co. Ltd.) with AgeI/EcoRI sites. Lentivirus particles

142 were prepared as previously described 23. RKO and HT-29 cells (2x105 cells/well)

143 were cultured in 6-well plates and transfected with either negative control (shCtrl)

144 lentivirus or KNL1 (shKNL1) lentivirus at a multiplicity of infection (MOI) of 5.

145 Cells were incubated for 5 days at 37˚C in an atmosphere containing 5% CO2. After

146 72 h of transfection, cells were observed using a ﬂuorescence microscope (IX71;

147 Olympus Corp., Tokyo, Japan). When the 5-day transfection was complete,

148 knockdown efficiency was measured using western blotting and RT-qPCR.

149 Tianliang Bai et al: Effect of KNL1 on the proliferation and apoptosis bioRxiv preprint doi: https://doi.org/10.1101/499889; this version posted December 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

150 Cell growth assay.

151 ShKNL1-transfected or shCtrl-transfected RKO and HT-29 cells in the logarithmic

152 growth phase were cultured in 96-well plates at a density of 2,000 cells per well for 5

153 days at 37˚C in an atmosphere containing 5% CO2. Cell numbers were recorded every

154 day using a Celigo Imaging Cytometer (Nexcelom Bioscience, Lawrence, MA, USA),

155 and all were performed in triplicate.

156

157 Methyl-thiazol-tetrazolium (MTT) assay.

158 ShKNL1-transfected or shCtrl-transfected RKO and HT-29 cells were again seeded

159 and cultured in 96-well plates at a density of 2000 cells/well at 37˚C. Cell

160 proliferation was assessed each day for 5 days. Brieﬂy, 20 µl MTT (5 mg/ml,

161 Genview, Craigie Burn, Australia) was added to each well and incubated at 37˚C for 4

162 h. The culture medium was removed and 100 µl of dimethyl sulfoxide was added to

163 each well to dissolve the crystals. Plates were agitated for 2-5 min and the absorbance

164 at 490 nm was read using a microplate reader (Tecan infinite, Tecan GmbH, Austria).

165 All experiments were performed in triplicate.

166

167 Colony formation assay

168 Cells transfected with shKNL1 or shCtrl vectors in the logarithmic growth phase were

169 re-suspended in RPMI-1640 and seeded in 6-well plates at a density of 800 cells/well.

170 Cells were incubated for 14 days and fluorescence microscopy (IX71; Olympus Corp.)

171 was used to observe cell colonies. Cells were fixed with paraformaldehyde (1 ml/well;

172 Sangon Biotech Co. Ltd., Shanghai, China) for 30 min. Cells were subsequently

173 washed with PBS and stained with 500 µl 0.5% crystal violet (Sangon Biotech Co.

174 Ltd., Shanghai, China) for 5 min. After the cellular staining, ddH2O was used to wash

175 the stained cells, which were then dried at room temperature. Finally, cell colonies

176 were observed under a microscope (Cai Kang Optical Instrument Co., Ltd, Shanghai,

177 China). All experiments were performed in triplicate.

178

179 FACS analysis. Fluorescence-activated cell sorting (FACS) was Tianliang Bai et al: Effect of KNL1 on the proliferation and apoptosis bioRxiv preprint doi: https://doi.org/10.1101/499889; this version posted December 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

180 used to analyze cell apoptosis24.

181 In brief, RKO cells transfected with shCtrl or shKNL1 were seeded in 6-well plates

182 for 120 h and grown to 70% conﬂuence, harvested and washed with 1X binding buffer,

183 following which 10 µl of Annexin V-allophycocyanin (APC) Detection kit

184 (eBioscience; Thermo Fisher Scientifc, Inc.) was added per 200 µl of the adjusted

185 suspension for 15 min in the dark at room temperature. Flow cytometry analysis after

186 cellular staining. All experiments were performed in triplicate.

187 Statistical analysis.

188 SPSS version 19.0 for Windows (IBM Corp., Armonk, NY, USA) was used for

189 statistical analysis and all data are presented as the mean ± standard deviation.

190 Comparisons between the two groups were made using Student's t-test and P<0.05

191 was considered to indicate a statistically significant difference.

192

193 Results

194 Compared with normal adjacent tissues, the expression of KNL1 was obviously

195 higher in colorectal tumor tissues.

196 RT-qPCR and Western blot were used to measure the expression levels of KNL1 in

197 CRC and para-cancerous tissues. The results revealed that, compared with normal

198 colorectal tissues, KNL1 mRNA and protein levels were much higher in CRC tissues

199 (Fig. 1A and B).

200

201 KNL1 expression in 4 colorectal cancer cell lines.

202 RT-qPCR and western blotting were used to measure the expression of KNL1 mRNA

203 and protein in SW480, RKO, HT-29, HCT-116 CRC cells and the normal colonic

204 epithelial cell line NCM460. The results indicated that KNL1 mRNA and protein was

205 highly expressed in in SW480, RKO, HT-29 and HCT-116 cell lines (Fig. 2).

206

207 Lentivirus-mediated knockdown of KNL1 in CRC cells.

208 To assess the mechanism of KNL1 in CRC, KNL1 expression was knocked down in

209 RKO and HT-29 cells using transfection. After 3 days, >80% of the cells were Tianliang Bai et al: Effect of KNL1 on the proliferation and apoptosis bioRxiv preprint doi: https://doi.org/10.1101/499889; this version posted December 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

210 successfully transfected with either an shCtrl or an shKNL1 lentivirus (Fig. 3A and D).

211 RT-qPCR was performed 5 days after transfection and KNL1 mRNA expression in the

212 shKNL1 group was much lower compared with in shCtrl-infected control cells (Fig.

213 3B and E). These results were confirmed using western blotting, and it was clear that

214 shKNL1-transfected cells had largely reduced KNL1 levels, indicating that the target

215 gene was successfully knocked down (Fig. 3C and F).

216

217 The CRC cell proliferation is inhibited by KNL1 knockdown.

218 RKO and HT-29 cells transfected with shCtrl or shKNL1 lentivirus were seeded into

219 96-well plates and analyzed using Celigo for 5 consecutive days. The number of cells

220 in the shCtrl group considerably increased over the 5 days, whereas the cell number in

221 the shKNL1-transduced group increased slightly (Fig. 4A-C and 4F-H). These results

222 suggest that KNL1 knockdown significantly inhibits CRC cell proliferation, which

223 was further confirmed by the MTT results (Fig. 4D-E and 4I-J). It appears that KNL1

224 expression determines the growth of RKO and HT-29 cells.

225

226 The formation of CRC cell colonies is inhibited by the KNL1 knockdown.

227 RKO and HT-29 cells were stained with crystal violet to assess colony formation (Fig.

228 5A and C). The number of the cells per colony was significantly higher in the shCtrl

229 group compared with the shKNL1 group (shCtrl: 229±9 vs. shKNL1: 40±5; shCtrl:

230 259±2 vs. shKNL1: 55±3; P<0.01) (Fig. 5B and D), indicating that the endogenous

231 reduction in KNL1 expression significantly inhibited CRC cell growth.

232

233 KNL1 knockdown affects the apoptosis of RKO cells.

234 The apoptotic rate of RKO cells in the shKNL1 group was 14.86±0.30%, whereas it

235 was 4.32±0.13% in the shCtrl group. The results suggest that apoptosis was

236 significantly increased in the shCtrl group compared with the shNKL1 group (P<0.05)

237 (Fig. 5E and F).

238

239 Discussion Tianliang Bai et al: Effect of KNL1 on the proliferation and apoptosis bioRxiv preprint doi: https://doi.org/10.1101/499889; this version posted December 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

240 The morbidity and mortality rate of CRC has risen rapidly, and it has attracted

241 worldwide attention 25, 26. Despite improvements in clinical practice and treatment,

242 outcomes to CRC patients are still poor due to metastasis or drug resistance 27, 28.

243 Therefore, it is necessary to determine new therapeutic targets and to better

244 understand the underlying molecular mechanism.

245 The occurrence and development of tumors are associated with both behavioral

246 and environmental factors, and tumorigenesis results from genetic mutations

247 accumulating over time. Identifying the affected genes may allow for the development

248 of advanced diagnostic and treatment methods for cancer.

249 The spindle checkpoint controls mitotic progression. The human kinetochore

250 oncoprotein AF15q14/KNL1, which belongs to the Spc105/Spc7/KNL-1 family, has

251 been reported to provide direct links between Bub1 and BubR1 spindle checkpoint

252 proteins and the kinetochores, and also plays a crucial role in the alignment of

253 chromosomes and spindle checkpoints. KNL1 RNAi gives induces accelerated

254 mitosis due to chromosome misalignment and checkpoint failure, which are caused by

255 the lack of microtubule attachment and kinetochores 16, 29. Another study reported that

256 transfection with KNL1 siRNA resulted in cancer cell apoptosis and inhibited the

257 growth of these cells in both in vitro and in vivo, regardless of p53 status was 30.

258 However, to the best of our knowledge, there have not previously been specific

259 studies regarding KNL1 expression in human cancers, particularly CRC.

260 The results of the present study suggest an obvious relationship between KNL1

261 and CRC cell apoptosis and proliferation. Although many studies described the

262 abnormal mitosis in cancer cells treated with KNL1 siRNA 16, 31, we are the first to

263 demonstrate that KNL1 downregulation induced apoptosis and inhibited the growth of

264 CRC cell lines. However, there were a number of limitations to consider. The number

265 of clinical samples was small and a larger sample size is required to verify the

266 expression of KNL1 in colorectal tumors. There was also a lack of relevant clinical

267 information regarding the study participants.

268 In conclusion, the results of the present study suggest that shRNA downregulates

269 the expression of KNL1 in CRC cells, which in turn induces apoptosis and inhibits Tianliang Bai et al: Effect of KNL1 on the proliferation and apoptosis bioRxiv preprint doi: https://doi.org/10.1101/499889; this version posted December 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

270 proliferation. KNL1 knockdown may therefore be an effective assumed therapeutic

271 approach for the treatment of CRC in which KNL1 is overexpressed.

272 Acknowledgement

273 Not applicable.

274 Conflict of interest

275 All authors declare that they have no conflict of interest.

276 Availability of data and materials

277 The datasets used and/or analyzed during the current study are available from the

278 corresponding author on reasonable request.

279

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356 Figure 1. Expression of KNL1 is upregulated in CRC tissues. The mRNA (A)

357 and protein (B) expression levels of KNL1 were significantly higher in CRC

358 tissues than in the corresponding noncancerous tissues.*p<0.05.

359 Figure 2. KNL1 mRNA and protein levels in four colorectal cell lines and the

360 normal colonic epithelial cell line. (A)KNL1 mRNA expression levels determined

361 by RT-qPCR. (B and C)KNL1 protein expression levels determined by western

362 blotting. **P <0.01.

363 Figure 3. Knockdown of KNL1 in RKO and HT-29 cells infected with

364 shKNL1 or shCtrl. (A and D) Cells were examined by ﬂuorescent and light

365 microscopy at day 3 post-infection (x200, magnification). Representative images

366 of the cultures are shown. (B and E) KNL1 mRNA levels were analyzed using

367 RT-qPCR at day 5 post-infection. KNL1 mRNA level decreased significantly

368 after KNL1 knockdown（**P<0.01）. (C and F) KNL1 protein expression was

369 analyzed by western blotting in the transduced RKO and HT-29 cells.

370 Figure 4. KNL1 knockdown inhibits RKO cell growth as compared with

371 control group. (A)RKO cells are infected by shCtrl and shKNL1 for 5 days, and

372 ﬂuorescence microscope (green staining) shows that the cell count is increased in

373 a time-dependent model. (B)Celigo Cell Counting indicated that cell growth is

374 slower in shKNL1 group than in shCtrl group. (C)The cell count and count fold

375 curves show significant differences between shKNL1 group and shCtrl group.

376 (D)The proliferative abilities of RKO cells transfected with shCtrl and shKNL1,

377 which were evaluated by MTT assay.(E)Downregulation of KNL1 remarkably

378 inhibited the proliferation rate of RKO cells as measured by the MTT assay.

379 KNL1 knockdown inhibits HT-29 cell growth as compared with control group.

380 (F)HT-29 cells are infected by shCtrl and shKNL1 for 5 days, and ﬂuorescence

381 microscope (green staining) shows that the cell count is increased in a

382 time-dependent model. (G)Celigo Cell Counting indicated that cell growth is

383 slower in shKNL1 group than in shCtrl group. (H)The cell count and count fold

384 curves show significant differences between shKNL1 group and shCtrl group. (I)The

385 proliferative abilities of HT-29 cells transfected with shCtrl and shKNL1, which were

386 evaluated by MTT assay.(J)Downregulation of KNL1 remarkably inhibited the

387 proliferation rate of HT-29 cells as measured by the MTT assay. **P <0.01.

388 Figure 5. KNL1 silencing represses RKO and HT-29 cells colony formation. (A

389 and C) Photomicrographs of crystal violet-stained colonies of RKO and HT-29 cells

390 growing in 6-well plates for 10 days after infection. (B and D)The number of cells in

391 each colony of RKO and HT-29 cells was counted. Cell number in shKNL1 group

392 was significantly fewer relative to shCtrl-transduced cells. RKO cell apoptosis

393 analyzed by ﬂow cytometry. (E)Cells are transfected with shKNL1 and shCtrl,

394 apoptosis is analyzed by ﬂow cytometry. Each group is shown in triplicates. (F)The

395 resulting data indicate that the cell apoptosis is significantly higher in shKNL1 group

396 than in shCtrl group. **P <0.01.

397

Tianliang Bai et al: Effect of KNL1 on the proliferation and apoptosis bioRxiv preprint doi: https://doi.org/10.1101/499889; this version posted December 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/499889; this version posted December 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/499889; this version posted December 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/499889; this version posted December 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/499889; this version posted December 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.