ライフサイエンスコーパス: C. burnetii

1 C. burnetii DeltacvpB, DeltacvpC, DeltacvpD, and Deltacv

2 C. burnetii extracts and rACP were also able to inhibit

3 C. burnetii has evolved to replicate in this harsh compa

4 C. burnetii infection affected the expression of multipl

5 C. burnetii infection of THP-1 human macrophage-like cel

6 C. burnetii infection prevented caspase 3/7 activation a

7 C. burnetii infects mammalian cells and then remodels th

8 C. burnetii isolates have a range of disease potentials;

9 C. burnetii lacks enzymes for de novo cholesterol biosyn

10 C. burnetii persists within and is transmitted by mammal

11 C. burnetii requires this lysosomal environment for repl

12 C. burnetii was coelectroporated with a plasmid encoding

13 C. burnetii-infected cells demonstrated significant prot

14 Over 100 C. burnetii Dot/Icm substrates have been identified, but

15 A C. burnetii DeltacvpA mutant exhibited significant defec

16 bility of PIV to confer protection against a C. burnetii infection.

17 Here, we constructed a C. burnetii pmrA deletion mutant to directly probe PmrA-

18 h rifampin, indicating their activation is a C. burnetii-directed event requiring pathogen RNA synthe

19 Using micromanipulation, a C. burnetii clone was isolated containing a Tn insertion

20 e here the cloning and characterization of a C. burnetii ftsZ mutant generated by mariner-based Himar

21 Apoptosis inhibition was a C. burnetii-driven process as infected cells treated wit

22 e) buffers have been described that activate C. burnetii metabolism in vitro, but metabolism is short

23 associated with the production of additional C. burnetii proteins involved in host cell parasitism.

24 We predict additional C. burnetii effectors localize to the PV membrane and re

25 d significant protection against aerosolized C. burnetii, suggesting that 1E4 may be useful for preve

26 he early immune response against aerosolized C. burnetii.

27 ction, SCID mice were exposed to aerosolized C. burnetii.

28 r C. burnetii 3262 stimulation but not after C. burnetii Nine Mile stimulation.

29 6 led to decreased cytokine production after C. burnetii 3262 stimulation but not after C. burnetii N

30 ay an important role in host defense against C. burnetii infection in mice.

31 r peritoneal B cells in host defense against C. burnetii infection in vivo.

32 e of neutrophils in the host defense against C. burnetii infection remains unclear.

33 mmunity is critical for host defense against C. burnetii infection.

34 ay an important role in host defense against C. burnetii pulmonary infection.

35 f neutrophils in protective immunity against C. burnetii infection, the RB6-8C5 antibody was used to

36 the mechanisms of pulmonary immunity against C. burnetii infection.

37 r neutrophils in protective immunity against C. burnetii infection.

38 nsfer of 1E4 would protect SCID mice against C. burnetii aerosol infection.

39 manized MAb as emergency prophylaxis against C. burnetii exposure.

40 ble to confer significant protection against C. burnetii challenge.

41 tical role in PIV-induced protection against C. burnetii infection.

42 ibodies (Abs) can provide protection against C. burnetii natural infection, we examined if passive tr

43 the induction of cytokine responses against C. burnetii in humans.

44 All C. burnetii isolates carry a large, autonomously replica

45 and plasmid ORFs were polymorphic among all C. burnetii isolates, representing ca. 7% of the NMI cod

46 smid, three of which are conserved among all C. burnetii isolates, suggesting that they are critical

47 oteins (CpeA, CpeB, and CpeF) encoded by all C. burnetii plasmids and IPS are Dot/Icm substrates.

48 open reading frame (CbuD1884) present in all C. burnetii isolates except the Nine Mile reference isol

49 This immune evasion strategy may allow C. burnetii to persist in an immunocompetent host.

50 We compared antigenic polypeptides among C. burnetii isolates and found an immunodominant 28-kDa

51 that neutrophils cannot kill C. burnetii and C. burnetii may be using infection of neutrophils as an

52 e positive for B. quintana, B. henselae, and C. burnetii, respectively, by the dPCR assay, which matc

53 ent recognition of C. burnetii Nine Mile and C. burnetii 3262.

54 their noted similarities, L. pneumophila and C. burnetii are exquisitely adapted for replication in u

55 needed for biogenesis of prototypical PV and C. burnetii replication.

56 The attenuated C. burnetii phase II (having a truncated "O" chain of it

57 infection in vitro and found that avirulent C. burnetii triggers sustained interleukin-1beta (IL-1be

58 Compared to wild-type bacteria, C. burnetii DeltapmrA exhibited severe intracellular gro

59 Moreover, correlations between C. burnetii genomic groups and human disease presentatio

60 sed on understanding the interaction between C. burnetii and innate immune cells in vitro and in vivo

61 Studying the interaction between C. burnetii and the innate immune system can provide a m

62 athogen's proteome, probed with biotinylated C. burnetii genomic DNA.

63 th Fab1E4, muscFv1E4, or huscFv1E4 can block C. burnetii infection of macrophages.

64 d substrates of the Dot/Icm system from both C. burnetii and L. pneumophila.

65 PMN were challenged with viable C. burnetii, C. burnetii extracts, or rACP but not when PMN were chal

66 It is likely that inhibition of apoptosis by C. burnetii represents an important virulence property t

67 The effector proteins delivered by C. burnetii are predicted to have important functions du

68 tic tools, secretion of plasmid effectors by C. burnetii during host cell infection was confirmed usi

69 e secreted in a Dot/Icm-dependent fashion by C. burnetii during infection of human THP-1 macrophages.

70 e vesicular trafficking pathways co-opted by C. burnetii for PV development are poorly defined; howev

71 ored in Escherichia coli deletion strains by C. burnetii recA or addAB.

72 olonged de novo protein and ATP synthesis by C. burnetii (>24 h).

73 kinase pathways are most likely targeted by C. burnetii Icm/Dot effectors.

74 with L. pneumophila was also translocated by C. burnetii in a process that requires its C terminus, p

75 In human cells, C. burnetii generates a replication niche termed the par

76 ining a valuable approach for characterizing C. burnetii interactions with a human host.

77 itive and specific option for characterizing C. burnetii isolates, especially when coupled with antig

78 Cloned C. burnetii fur complemented an Escherichia coli fur del

79 eal B cells alone may not be able to control C. burnetii infection.

80 ion of Akt or Erk1/2 significantly decreased C. burnetii antiapoptotic activity.

81 Various formats using different C. burnetii antigens were tested.

82 effector that influences ER function during C. burnetii infection.

83 ctivity, no p62 turnover was observed during C. burnetii growth in macrophages, suggesting that the p

84 es were differentially phosphorylated during C. burnetii infection, suggesting the pathogen uses PKA

85 and probed the role of PKA signaling during C. burnetii infection of macrophages.

86 st the enzyme may act on host sterols during C. burnetii intracellular growth.

87 Vero cells were infected with electroporated C. burnetii and transformants scored as organisms replic

88 In all cell types examined, C. burnetii establishes a replicative niche in a lysosom

89 is, lytic cell death did not occur following C. burnetii-triggered inflammasome activation, indicatin

90 he absence of LexA, co-protease activity for C. burnetii RecA was demonstrated.

91 To compensate for C. burnetii auxotrophies and other potential metabolic d

92 Several effectors crucial for C. burnetii intracellular replication have been identifi

93 tion of HeLa cells, which are permissive for C. burnetii replication.

94 iological buffers (pH 4.5) were screened for C. burnetii metabolic permissiveness.

95 sehold contacts were traced and screened for C. burnetii.

96 generation of human granulomas specific for C. burnetii.

97 gest the value of systematically testing for C. burnetii in antiphospholipid-associated cardiac valve

98 In all assay formats, C. burnetii-specific IFN-gamma production was higher (P

99 involved in protecting vaccinated mice from C. burnetii challenge-induced disease.

100 antigen A) gene was detected in acute group C. burnetii isolates but not identified in chronic group

101 Importantly, axenically grown C. burnetii were highly infectious for Vero cells and ex

102 xenic medium that supports sustained (>24 h) C. burnetii metabolic activity.

103 However, significantly higher C. burnetii genome copy numbers were detected in the lun

104 However, we do not understand how C. burnetii evades the intracellular immune surveillance

105 To understand how C. burnetii maintains genomic integrity in this environm

106 In humans, C. burnetii infects alveolar macrophages and promotes ph

107 ological responses to infection with phase I C. burnetii isolates from the following four genomic gro

108 ed by using (i) a genetic screen to identify C. burnetii proteins interacting with DotF, a component

109 oring virulent phase I or avirulent phase II C. burnetii variants in human mononuclear phagocytes.

110 Inhibition of apoptosis in C. burnetii-infected cells did not correlate with the de

111 Specific biomarkers were detected in C. burnetii Nine Mile phase I (NMI) strain purified from

112 itical role for extrachromosomal elements in C. burnetii pathogenesis.

113 The presence of three selfish elements in C. burnetii's 23S rRNA gene is very unusual for an oblig

114 Expression of a subset of repair genes in C. burnetii was monitored and, in contrast to the non-in

115 results suggest a versatile role for PKA in C. burnetii infection and indicate virulent organisms us

116 ws the expression of recombinant proteins in C. burnetii as TEM fusion products.

117 tism and opens the door for a renaissance in C. burnetii research.

118 d ERK led to decreased cytokine responses in C. burnetii-stimulated human PBMCs.

119 the fragmented nature of mature 23S rRNA in C. burnetii due to the presence of an intervening sequen

120 ested a very limited Fur-regulated system in C. burnetii.

121 ect genetic evidence of a functional T4SS in C. burnetii.

122 ce of clathrin-coated vesicle trafficking in C. burnetii infection and define a role for CvpA in subv

123 re challenged with electron beam-inactivated C. burnetii.

124 Based on [(35)S]Cys-Met incorporation, C. burnetii displayed optimal metabolic activity in citr

125 Collectively, these data indicate C. burnetii encodes multiple effector proteins that targ

126 uscFv1E4, and huscFv1E4 were able to inhibit C. burnetii infection in mice but that their ability to

127 accinated WT mouse sera were able to inhibit C. burnetii infection in vivo, but only IgM from PIV-vac

128 esults indicate that 1E4 was able to inhibit C. burnetii infection in vivo, suggesting that 1E4 is a

129 41920-KLH-immunized mice was able to inhibit C. burnetii infection in vivo, suggesting that m1E41920

130 on in mice but that their ability to inhibit C. burnetii infection was lower than that of 1E4.

131 v1E4) retained the ability of 1E4 to inhibit C. burnetii infection.

132 CD4(+) T cell-deficient mouse sera inhibited C. burnetii infection.

133 l antibody (MAb) 1E4 significantly inhibited C. burnetii infection in mice, suggesting that 1E4 is a

134 n by siRNA treatment significantly inhibited C. burnetii replication.

135 Instead, C. burnetii possesses addAB orthologous genes, functiona

136 Interestingly, C. burnetii inside neutrophils can infect and replicate

137 the severity of disease following intranasal C. burnetii challenge, suggesting that keratinocyte-deri

138 n the current study, we further investigated C. burnetii manipulation of host cell signaling and apop

139 Human DC were infected with two isogenic C. burnetii strains that differ in LPS length.

140 tii Nine Mile and the Dutch outbreak isolate C. burnetii 3262.

141 ges, suggesting that neutrophils cannot kill C. burnetii and C. burnetii may be using infection of ne

142 ent dotA mutants trafficked to lysosome-like C. burnetii vacuoles in Vero cells where they survived b

143 In human alveolar macrophages, C. burnetii uses a Dot/Icm type IV secretion system (T4S

144 he lack of methods to genetically manipulate C. burnetii significantly impedes the study of this orga

145 human dendritic cells (DC) containing mature C. burnetii replication vacuoles were superinfected with

146 n tissue culture host cells or axenic media, C. burnetii extracts, or purified recombinant ACP (rACP)

147 The low rate of phase I and II Nine Mile C. burnetii growth in murine lungs may be a direct resul

148 doses of both phase I and phase II Nine Mile C. burnetii multiply and are less readily cleared from t

149 Moreover, C. burnetii morphological differentiation was induced in

150 gments of an LPS-specific MAb can neutralize C. burnetii infection and appears to be a promising step

151 Our data reveal IcaA as a novel C. burnetii effector protein that is secreted by the Dot

152 mophila as a surrogate host, reveals a novel C. burnetii gene (IcaA) involved in the inhibition of ca

153 To identify novel C. burnetii effectors, we applied a machine-learning app

154 stantial growth (approximately 3 log(10)) of C. burnetii in a 2.5% oxygen environment.

155 he current study, we examined the ability of C. burnetii to inhibit apoptotic cell death during infec

156 The sustained in vitro metabolic activity of C. burnetii in CCM provides an important tool to investi

157 genome reduction suggests the adaptation of C. burnetii to an obligate intracellular lifestyle is a

158 likely reflect a unique repair adaptation of C. burnetii to its hostile niche.

159 oaerosols via the air, the aerosolization of C. burnetii in the shower, and the air filtration effici

160 In this study a proteome analysis of C. burnetii developmental forms was conducted to provide

161 t IL-1 may be important for the clearance of C. burnetii from the lungs following intranasal infectio

162 t that T cells are critical for clearance of C. burnetii, either NM I or NM II, that IFN-gamma and TN

163 for both RNI and ROI in the host control of C. burnetii infection.

164 Axenic cultivation of C. burnetii will facilitate studies of the organism's pa

165 s were quantified in synchronous cultures of C. burnetii and found to closely parallel those of 16S r

166 genomes throughout a 14-day growth cycle of C. burnetii and found that they were inversely correlate

167 This is the first description of C. burnetii harboring a defined gene mutation generated

168 le for TNF produced upon immune detection of C. burnetii NMII by TLRs, rather than cytosolic PRRs, in

169 prehensively define the genetic diversity of C. burnetii by hybridizing the genomes of 20 RFLP-groupe

170 prior to challenge with the highest dose of C. burnetii were protected against lethal infection and

171 ing and apoptosis by examining the effect of C. burnetii infection on activation of 15 host proteins

172 bserved and more apparent with expression of C. burnetii RecAG159D mutant protein.

173 ring), respectively, in the 23S rRNA gene of C. burnetii.

174 The ?2 Mb genome of C. burnetii is about twice the size of genomes of most o

175 Distinct genomic groups of C. burnetii are revealed by restriction fragment-length

176 strains belonging to eight genomic groups of C. burnetii to determine sequence variation and the pres

177 wth and PV generation, whereas the growth of C. burnetii DeltacvpB and DeltacvpC was rescued upon coh

178 ly, these results indicate the importance of C. burnetii modulation of host signaling and demonstrate

179 Dominant-negative inhibition of C. burnetii RecA by recA mutant alleles, modelled after

180 Here, we characterized the interaction of C. burnetii with dendritic cells (DC), critical componen

181 The mechanisms of C. burnetii intracellular survival are poorly defined an

182 Moreover, icaA(-) mutants of C. burnetii failed to suppress the caspase-11-mediated i

183 The persistent and mild nature of C. burnetii infection in vitro suggests that the pathoge

184 variant (NMI) or phase II variant (NMII) of C. burnetii.

185 a T4SS are implicated in the pathogenesis of C. burnetii in flies.

186 characterized a thiol-specific peroxidase of C. burnetii that belongs to the atypical 2-cysteine subf

187 viously described immunodominant proteins of C. burnetii and novel immunogenic proteins that may be i

188 , genomic rearrangements, and pseudogenes of C. burnetii isolates are consistent with genome structur

189 hypothesize that inefficient recognition of C. burnetii and/or activation of host-defense in individ

190 known about their role in the recognition of C. burnetii in humans.

191 ing finding was the divergent recognition of C. burnetii Nine Mile and C. burnetii 3262.

192 with a known role in initial recognition of C. burnetii were included.

193 Replication of C. burnetii during infection has been shown to be increa

194 at restrict the intracellular replication of C. burnetii.

195 expression profiling, allowed the rescue of C. burnetii from its host cell to regain the axenic grow

196 is factor (TNF) produced upon TLR sensing of C. burnetii NMII was required to control bacterial repli

197 did not significantly affect the severity of C. burnetii infection-induced diseases in both severe co

198 Nine Mile phase II (NMII) clone 4 strain of C. burnetii, as a model to investigate host and bacteria

199 sing the Nine Mile phase II (NMII) strain of C. burnetii.

200 that adaA is exposed on the outer surface of C. burnetii.

201 ar (pppy), hence the risk of transmission of C. burnetii through inhalation of drinking water aerosol

202 s are the aeration process, the transport of C. burnetii in bioaerosols via the air, the aerosolizati

203 In addition, treatment of C. burnetii with Fab1E4, muscFv1E4, or huscFv1E4 can blo

204 Interestingly, treatment of C. burnetii with huscFv1E4 can significantly reduce C. b

205 To provide a more-complete understanding of C. burnetii's genetic diversity, evolution, and pathogen

206 sms in TLRs and Nod-like receptors (NLRs) on C. burnetii-induced cytokine production was assessed.

207 Indeed, insight from early work on C. burnetii metabolism, along with new information gaine

208 Optimal C. burnetii metabolism occurred in CCM with a high chlor

209 r family equips transmissive L. pneumophila, C. burnetii, and F. tularensis to assess their phagosoma

210 close association with acidic, LAMP-positive C. burnetii replication vacuoles.

211 plied a machine-learning approach to predict C. burnetii effectors, and examination of 20 such protei

212 uolar effector proteins, a list of predicted C. burnetii T4BSS substrates was compiled using bioinfor

213 ossibility of using humanized 1E4 to prevent C. burnetii infection, we examined whether the Fab fragm

214 esting that 1E4 may be useful for preventing C. burnetii natural infection.

215 ion is a novel diagnostic assay for previous C. burnetii infection and shows similar performance and

216 e of B cells in host defense against primary C. burnetii infection remains unclear.

217 portant role in host defense against primary C. burnetii infection.

218 portant role in host defense against primary C. burnetii infection.

219 d effector protein CvpA was found to promote C. burnetii intracellular growth and PV expansion.

220 ne responses in the lung following pulmonary C. burnetii infection is lacking.

221 etii with huscFv1E4 can significantly reduce C. burnetii infectivity in human macrophages.

222 s a critical virulence factor that regulates C. burnetii Dot/Icm secretion.

223 blotting using two-dimensional gel-separated C. burnetii antigens.

224 To study C. burnetii phase II infections, febrile responses in ga

225 In this study, C. burnetii-specific interferon gamma (IFN-gamma) produc

226 To define conditions that support C. burnetii growth, we systematically evaluated the orga

227 Formation of a PV that supports C. burnetii replication requires a Dot/Icm type 4B secre

228 Coxiella-containing vacuoles (CCVs) and that C. burnetii can infect and replicate in peritoneal B1a s

229 Together, these results demonstrate that C. burnetii actively directs PV-autophagosome interactio

230 thesis reduced the antiapoptotic effect that C. burnetii exerted on infected host cells.

231 In contrast, our finding that C. burnetii infection induced more-severe splenomegaly a

232 These data indicate that C. burnetii can interfere with the intrinsic cell death

233 Collectively, these results indicate that C. burnetii encodes a large repertoire of T4SS substrate

234 Recent studies have indicated that C. burnetii likely originated from a tick-associated anc

235 We recently reported that C. burnetii inhibits apoptotic cell death in macrophages

236 Here, we show that C. burnetii inhibits caspase-1 activation in primary mou

237 Recent reports suggest that C. burnetii actively recruits autophagosomes to the PV t

238 These data suggest that C. burnetii does not actively inhibit phagolysosome func

239 Collectively, these data suggest that C. burnetii isolates translocate distinct subsets of the

240 Collectively, these data suggest that C. burnetii modulates apoptotic pathways to inhibit host

241 Collectively, these results suggest that C. burnetii plasmid-encoded T4SS substrates play importa

242 Earlier studies suggested that C. burnetii actively inhibited release of ROI from PMN t

243 n rates were lower than 29%, suggesting that C. burnetii can infect neutrophils, but infection is lim

244 The C. burnetii NMII T4SS translocates bacterial products in

245 of genetic systems, protein transfer by the C. burnetii Dot/Icm has not been demonstrated.

246 s, translocation of effector proteins by the C. burnetii Dot/Icm system occurs after acidification of

247 the T4SS effector repertoire encoded by the C. burnetii QpH1, QpRS, and QpDG plasmids that were orig

248 on and the engagement of this pathway by the C. burnetii type 4B secretion system substrate Coxiella

249 ysis revealed multiple transpositions in the C. burnetii genome and rescue cloning identified 30 and

250 may play an important role in inhibiting the C. burnetii infection-induced inflammatory response.

251 tion vacuoles, and factors that maintain the C. burnetii replication vacuole do not alter biogenesis

252 t the identification of 32 substrates of the C. burnetii Dot/Icm system using a fluorescence-based be

253 ere identified upon genome sequencing of the C. burnetii Nine Mile reference isolate, which is associ

254 a similar role for PmrA in regulation of the C. burnetii T4BSS has been proposed.

255 estrict the intracellular replication of the C. burnetii.

256 coding genes were recently discovered on the C. burnetii cryptic QpH1 plasmid, three of which are con

257 Here we show that the C. burnetii secreted effector Coxiella vacuolar protein

258 Within whole lung tissue, C. burnetii preferentially replicated in hAMs.

259 first evidence of exposure of polar bears to C. burnetii, N. caninum, and F. tularensis.

260 scular risk-factors and previous exposure to C. burnetii.

261 have a beta-lactamase enzyme (BlaM) fused to C. burnetii effector proteins to study protein transloca

262 critical role in vaccine-induced immunity to C. burnetii infection by producing protective antibodies

263 udies suggested that Ab-mediated immunity to C. burnetii phase I LPS (PI-LPS) is protective.

264 vel of interleukin-10 (IL-10) in response to C. burnetii infection in vitro suggest that B1a cells ma

265 not play essential roles in the response to C. burnetii infection.

266 mechanisms of the innate immune response to C. burnetii natural infection, SCID mice were exposed to

267 studying the human innate immune response to C. burnetii.

268 TLR1, increased interleukin 10 responses to C. burnetii in individuals carrying the risk allele may

269 at adult Drosophila flies are susceptible to C. burnetii NMII infection and that this bacterial strai

270 se functions, some of which may be unique to C. burnetii pathotypes.

271 Monocytes migrated toward C. burnetii-coated beads independently of the presence o

272 Following aerosol-mediated transmission, C. burnetii replicates in alveolar macrophages in a uniq

273 acterial cell mass spectrometry of wild-type C. burnetii and the DeltapmrA mutant uncovered new compo

274 We show that a unique C. burnetii effector from the ankyrin repeat (Ank) famil

275 ecrosis factor-alpha and interleukin-12 upon C. burnetii infection.

276 aerosol challenge model was developed using C. burnetii Nine Mile phase I (RSA 493), administered us

277 To test this model, viable C. burnetii propagated in tissue culture host cells or a

278 hibited when PMN were challenged with viable C. burnetii, C. burnetii extracts, or rACP but not when

279 Both virulent C. burnetii Nine Mile phase I (NMI) and avirulent Nine M

280 s also required for PV formation by virulent C. burnetii isolates during infection of primary human a

281 We propose a model whereby LPS of virulent C. burnetii masks toll-like receptor ligands from innate

282 al B cells were able to phagocytose virulent C. burnetii bacteria and form Coxiella-containing vacuol

283 To gain insight into the mechanisms by which C. burnetii is able to multiply intracellularly, we exam

284 These data support a model in which C. burnetii eludes the primary ROI killing mechanism of

285 creation of the specialized vacuole in which C. burnetii replicates represents a two-stage process me

286 While C. burnetii genomes are highly syntenous, recombination

287 ter in vitro stimulation of whole blood with C. burnetii antigens.

288 ssively accumulated around beads coated with C. burnetii extracts, and complete granulomas were gener

289 of these proteins increased coincident with C. burnetii replication through at least 72 hpi.

290 Whole blood incubated for 24 hours with C. burnetii Nine Mile showed optimal performance.

291 data show that mammalian cells infected with C. burnetii are resistant to apoptosis induced by stauro

292 ondria was diminished in cells infected with C. burnetii upon induction of apoptosis.

293 e with cardiac valve disease, infection with C. burnetii can cause a life-threatening infective endoc

294 We report on evidence of infection with C. burnetii in a small group of regular consumers of raw

295 ils in mice before intranasal infection with C. burnetii.

296 Following inoculation of the lungs with C. burnetii Nine Mile phase I (NMI), SCID mice developed

297 rmine that the infection of macrophages with C. burnetii inhibits the caspase-11-mediated non-canonic

298 on of IFN-gamma(-/-) and TLR2(-/-) mice with C. burnetii NMII 30 days after primary infection protect

299 wed increased interleukin 10 production with C. burnetii exposure.

300 nonuclear cells (PBMCs) were stimulated with C. burnetii Nine Mile and the Dutch outbreak isolate C.