Molecular Characterization of Resistance to Nicosulfuron in Setaria viridis.

The green foxtail, Setaria viridis (L.) P. Beauv. (Poales: Poaceae), is a troublesome and widespread grass weed in China. The acetolactate synthase (ALS)-inhibiting herbicide nicosulfuron has been intensively used to manage S. viridis, and this has substantially increased the selection pressure. Here we confirmed a 35.8-fold resistance to nicosulfuron in an S. viridis population (R376 population) from China and characterized the resistance mechanism. Molecular analyses revealed an Asp-376-Glu mutation of the ALS gene in the R376 population. The participation of metabolic resistance in the R376 population was proved by cytochrome P450 monooxygenases (P450) inhibitor pre-treatment and metabolism experiments. To further elucidate the mechanism of metabolic resistance, eighteen genes that could be related to the metabolism of nicosulfuron were obtained bythe RNA sequencing. The results of quantitative real-time PCR validation indicated that three ATP-binding cassette (ABC) transporters (ABE2, ABC15, and ABC15-2), four P450 (C76C2, CYOS, C78A5, and C81Q32), and two UDP-glucosyltransferase (UGT) (UGT13248 and UGT73C3), and one glutathione S-transferases (GST) (GST3) were the major candidates that contributed to metabolic nicosulfuron resistance in S. viridis. However, the specific role of these ten genes in metabolic resistance requires more research. Collectively, ALS gene mutations and enhanced metabolism may be responsible for the resistance of R376 to nicosulfuron.

EST-SSR Markers' Development Based on RNA-Sequencing and Their Application in Population Genetic Structure and Diversity Analysis of Eleusine indica in China.

Goosegrass (Eleusine indica) is one of the worst agricultural weeds in China. Molecular markers were developed for genetic diversity and population structure analyses. In this study, we identified 8391 expressed sequence tag-simple sequence repeat (EST-SSR) markers from the de novo assembled unigenes of E. indica. Mononucleotides were the most abundant type of repeats (3591, 42.79%), followed by trinucleotides (3162, 37.68%). The most dominant mononucleotide and trinucleotide repeat motifs were A/T (3406, 40.59%) and AAT/ATT (103, 1.5%), respectively. Fourteen pairs of EST-SSR primers were verified and used to analyze the genetic diversity and population structure of 59 goosegrass populations. A total of 49 alleles were amplified, with the number of alleles (Na) ranging from two to eleven per locus, and the effective number of alleles (Ne) ranged from 1.07 to 4.53. The average polymorphic information content (PIC) was 0.36. Genetic structure analysis (K = 2) and principal coordinate analysis divided 59 E. indica populations into two groups in a manner similar to the unweighted pair-group method (Dice genetic similarity coefficient = 0.700). This study developed a set of EST-SSR markers in E. indica and successfully analyzed the diversity and population genetic structures of 59 E. indica populations in China.

Multiple resistance to ACCase- and ALS-inhibiting herbicides in black-grass (Alopecurus myosuroides Huds.) in China.

Two black-grass (Alopecurus myosuroides Huds.) populations (R2105 and R1027) that were suspected to be resistant to clodinafop-propargyl, an acetyl-CoAcarboxylase (ACCase) inhibitor, were found in winter wheat fields in China. Research was carried out to investigate whether resistance to clodinafop-propargyl was present and the molecular mechanism of herbicide resistance in these two populations. Dose-response assays confirmed high level resistance to clodinafop-propargyl in both R2105 and R1027 populations, with resistance indexes 25.1 and 22.1. ACCase gene sequence comparison revealed three amino acid mutations (Trp-1999-Leu, Ile-2041-Asn, or Asp-2078-Gly) in R2105 population and Ile-2041-Asn mutation in R1027 population. Sensitivity to other herbicides assays indicated that R2105 and R1027 populations were cross resistant to fenoxaprop-P-ethyl and multiple resistant to pyroxsulam and mesosulfuron-methyl. The ALS gene sequence analysis revealed that all resistant individuals in R2105 and R1027 populations had the Trp-574-Leu mutation. Applying malathion, significantly decreased the rate of metabolism of clodinafop-propargyl in both R2105 and R1027 populations. This is the first report of multiple resistance to ACCase- and ALS-inhibiting herbicides conferred by target-site mutations and enhanced metabolism in black-grass in China.

Target gene mutation and enhanced metabolism confer fomesafen resistance in an Amaranthus retroflexus L. population from China.

Amaranthus retroflexus L., a troublesome annual dicotyledonous weed species, is highly competitive with soybean (Glycine max L.). A single-dose herbicide-resistance screening assay identified an A. retroflexus population with suspected resistance to fomesafen. Whole-plant dose-response assays demonstrated that the resistant population (2492) was resistant to protoporphyrinogen oxidase (PPO)-inhibiting herbicides (50.6-fold fomesafen resistance and > 8.1-fold lactofen resistance) compared to a susceptible (S) population. PPX2 gene sequence analysis showed an Arg128Gly amino acid substitution in the 2492 population. Moreover, pretreatment of malathion and the fomesafen metabolic assays through HPLC-MS demonstrated enhanced fomesafen metabolism in the 2492 population. Additionally, the 2492 population was 10.4-fold more resistant to the ALS-inhibiting herbicide imazethapyr and 16.8-fold more resistant to thifensulfuron-methyl than the S population. ALS gene sequence analysis showed an Ala205Val amino acid substitution in the 2492 population. This population of A. retroflexus has coexisting target-site resistance and non-target-site mechanisms for resistance to fomesafen. Multiple herbicide resistance may mean it is necessary to adjust weed management strategies to better control the resistant population.

Multiple resistance to ALS-inhibiting and PPO-inhibiting herbicides in Chenopodium album L. from China.

Common lambsquarters (Chenopodium album L.) is a broadleaf weed that can severely damage soybean fields. Two C. album populations (1744 and 1731) suspected resistant to imazethapyr were investigated for resistance levels to imazethapyr, thifensulfuron-methyl, and fomesafen and their resistance mechanisms were investigated. Whole-plant dose-response assays revealed that, compared to the susceptible (S) population, the 1744 population was 16.5-fold resistant to imazethapyr, slightly resistant to thifensulfuron-methyl (resistance index [R/S], <3). The 1731 population was 18.8-fold resistant to imazethapyr, 2.9-fold resistant to thifensulfuron-methyl, and 5.1-fold resistant to fomesafen. In vitro acetolactate synthase (ALS) assays showed 17.1-fold and 19.3-fold resistance levels of 1744 and 1731 populations to imazethapyr respectively. ALS gene sequence analysis identified Ala122Thr amino acid substitution in the 1744 population and Ser653Thr amino acid substitution in the 1731 population. No mutations of the protoporphyrinogen oxidase (PPO) gene were detected. However, pre-treatment with malathion reversed fomesafen resistance, suggesting nontarget-site resistance mechanisms likely play a role in the 1731 population.

Investigation of resistance mechanisms to bentazone in multiple resistant Amaranthus retroflexus populations.

Redroot amaranth (Amaranthus retroflexus L.) is a noxious weed that affects soybean production in China. Experiments were conducted to determine the molecular basis of resistance to bentazone. Whole-plant dose-response experiments showed that two populations (R1 and R2) exhibited resistance to bentazone with resistance indices of 9.01 and 6.85, respectively. Sequencing of the psbA gene revealed no amino acid substitution in the two populations. qRT-PCR analysis verified that psbA gene expression in R1 and R2 populations was increased significantly after treatment with bentazone, which was 3-fold and 5-fold higher than that in S1 and S2 populations, respectively. The P450 inhibitor malathion significantly reduced the level of resistance in the R1 and R2 populations when used prior to bentazone treatment. The R1 population exhibited multiple resistance to thifensulfuron-methyl and lactofen, caused by target site mutations (Asp-376-Glu in ALS, Arg-128-Gly in PPO2). In conclusion, increased gene expression of the psbA gene and enhanced herbicide metabolism seem to be the basis of resistance to bentazone in these A. retroflexus populations.

Autophagy Inhibition by ATG3 Knockdown Remits Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury and Inflammation in Brain Microvascular Endothelial Cells.

Autophagy participates in the development of cerebral ischemia stroke. Autophagy-related 3 (ATG3), an important autophagy regulator, was reported to be upregulated in a rat model of cerebral ischemia/reperfusion (CI/R) injury and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model. However, the detailed role of ATG3 in CI/R injury remains elusive. An in vitro cellular model was established to mimic CI/R injury by exposing hBMECs and bEnd.3 cells to OGD/R. OGD/R-induced injury were evaluated by cell counting kit-8 (CCK-8), LDH release assay, caspase-3 activity assay and TUNEL assay. Inflammation was assessed by detecting mRNA expression and concentrations of interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α) using qRT-PCR and ELISA, respectively. The protein levels of ATG3, light chain 3 (LC3)-I, LC3-II, p62, protein kinase B (Akt), and phosphorylated Akt (p-Akt) were determined by western blot analysis. We successfully established an in vitro OGD/R injury model using hBMECs and bEnd.3 cells. ATG3 was time-dependently upregulated and ATG3 knockdown inhibited autophagy in OGD/R-challenged brain microvascular endothelial cells. Moreover, autophagy inhibition by ATG3 interference attenuated OGD/R-induced viability inhibition and increase of LDH release, caspase-3 activity, programmed cell death, and production of IL-1ß, IL-6 and TNF-α. Inhibition of autophagy by ATG3 silencing activated the phosphoinositide 3-kinase (PI3K)/Akt pathway in OGD/R-challenged brain microvascular endothelial cells. Furthermore, inhibition of the PI3K/Akt pathway reversed the protective effects of ATG3 silencing on OGD/R-induced injury and inflammation. In conclusion, autophagy inhibition by ATG3 knockdown remitted OGD/R-induced injury and inflammation in brain microvascular endothelial cells via activation of the PI3K/Akt pathway.

Characterization of glyphosate and quizalofop-p-ethyl multiple resistance in Eleusine indica.

Glyphosate and Acetyl-coenzyme A Carboxylase (ACCase) inhibitors are popular herbicides that control goosegrass. However, some populations are difficult to control due to resistance resulting from the increasing selection pressure. The objectives of this research were to detect the multiple resistance levels, resistance mechanisms, and fitness costs of two goosegrass populations collected in China. The resistance indices of two resistant populations (denominated as R1 and R2) to glyphosate were 3.8 and 2.3, respectively; and it was 18.0 and 14.2 to quizalofop-p-ethyl, respectively. Shikimate accumulation in R1 and R2 populations was only 8% of that of the susceptible population after glyphosate treatment. A Pro-106-Ala mutation in EPSPS and an Asp-2078-Gly mutation in ACCase were present in both resistant populations. Both the expression level of EPSPS and ACCase in resistant populations were similar to that of susceptible populations. The leaf area of the individuals in wild-type populations was more than three times of the leaf area in the resistant populations. Similarly, resistant plants were 45-49% shorter, had 70-76% less fresh shoot weight, and 67-69% fewer seeds than wild-type plants. Goosegrass populations have evolved multiple resistance to glyphosate and the ACCase inhibitor quizalofop-p-ethyl in China. The Pro-106-Ala mutation in the EPSPS and the Asp-2078-Gly mutation in the ACCase were responsible for this resistance. In addition, a fitness cost exists in the resistant populations, and more work should conduct to clear which mutation is responsible for the fitness penalty.

Target site mutations and cytochrome P450s-involved metabolism confer resistance to nicosulfuron in green foxtail (Setaria viridis).

Green foxtail [Setaria viridis (L.) P.Beauv.] is a troublesome grass weed that is widely distributed in maize (Zea mays L.) fields across China. Many populations of S. viridis have evolved resistance to the acetolactate synthase (ALS)-inhibiting herbicide nicosulfuron. The objectives of this research were to confirm nicosulfuron resistance in these populations and to investigate the basis of nicosulfuron resistance. Whole-plant dose-response experiments showed 6 out of 13 S. viridis populations were highly resistance (20-30 times) to nicosulfuron. Sequencing of the ALS gene revealed two amino acid mutations, Asp-376-Glu and Pro-197-Ala, in the nicosulfuron-resistant populations. A malathion pretreatment study revealed that the R376 and R197 subpopulations might have cytochrome P450s-mediated herbicide metabolic resistance. The resistant populations were cross-resistant to imazethapyr but sensitive to topramezone and quizalofop-p-ethyl. This is the first report of resistance to ALS inhibitors conferred by target site mutations (Asp-376-Glu or Pro-197-Ser) and possible cytochrome P450s-involved metabolism in S. viridis.

Glyphosate resistance in Eleusine indica: EPSPS overexpression and P106A mutation evolved in the same individuals.

Goosegrass is one of the most widespread weeds in orchards and tea plantations in China, and glyphosate is a popular herbicide used to control it. However, high glyphosate selection pressure has led to some populations becoming resistant. The objectives of this research were to determine resistance levels and possible resistance mechanisms of goosegrass populations from several tea plantations in Zhejiang Province in China. The resistance indexes in four goosegrass populations (SH, SY, CA and CX) ranged from 4.9 to 13.4, and lower shikimate accumulation in these populations compared with a glyphosate-susceptible (GS) population confirmed their resistance to glyphosate. No mutations in the target gene EPSPS were found in populations SH and SY, however, the expression of EPSPS in these two populations was 9.3 and 29.7 times higher than that in the GS population, respectively. In the CX population, a P106S mutation in EPSPS was found in 6.7% of the individuals and another 80.0% of individuals had EPSPS amplification. In population CA, all the individuals had a P106A mutation and 86.7% of them had amplification in EPSPS. The EPSPS copy numbers ranged from 5.2 to 62.3 in these four resistant populations. There was a positive correlation between signal intensities of primary anti-EPSPS antibody and the copy number of the EPSPS protein, as indicated by immunoblot analysis. In population CA, with high-level resistance to glyphosate, both P106A mutation and amplification in EPSPS evolved in the same individuals in this population.

Investigation of resistance mechanism to fomesafen in Amaranthus retroflexus L.

Amaranthus retroflexus L. is one of the most troublesome weeds in autumn-crop fields in Northeast China. In recent years, field applications of fomesafen have failed to control an A. retroflexus population in Heilongjiang Province, China. Therefore, in this study, experiments were conducted to determine the resistance of A. retroflexus to fomesafen and investigate the molecular basis of herbicide resistance. Whole-plant dose-response experiments showed that the resistant (R) population exhibited 41.8-fold resistance to fomesafen compared with the susceptible (S) population. Target-gene sequence analysis revealed an Arg-128-Gly substitution in the protoporphyrinogen oxidase (PPO) in the R population. The response of PPO2 transgenic Arabidopsis thaliana to fomesafen demonstrated that the Arg-128-Gly substitution conferred high resistance to fomesafen. Cross- and multiple-resistance analyses indicated that the R population was cross-resistant to lactofen and carfentrazone-ethyl but was sensitive to imazethapyr, thifensulfuron-methyl, atrazine, and glyphosate. This study indicated that the Arg-128-Gly substitution is the main reason for A. retroflexus resistance to fomesafen. To our knowledge, this is the first report of a target-site based mechanism for the resistance to a PPO-inhibiting herbicide in A. retroflexus.

The basis of resistance mechanism to mesosulfuron-methyl in Tausch's goatgrass (Aegilops tauschii Coss.).

Tausch's goatgrass (Aegilops tauschii Coss.) is one of the most troublesome weeds in winter wheat-growing regions of China. In recent years, the recommended field rate of mesosulfuron-methyl failed to control the Tausch's goatgrass population in Shanxi province (SX), China. Experiments were conducted to characterize the herbicide resistance level and investigate the basis of mesosulfuron-methyl resistance in Tausch's goatgrass. Whole-plant dose-response tests showed that the SX population exhibited 11.42-fold resistance to mesosulfuron-methyl than the susceptible HN population, and the resistance level in the SX population could be significantly reduced by malathion, a cytochrome P450 inhibitor. The SX population also exhibited cross-resistance to imazethapyr, pyroxsulam and bispyribac­sodium. Acetohydroxyacid synthase (AHAS) sequencing and enzyme activity assays demonstrated that the mesosulfuron-methyl resistance was not conferred by target-site substitution. A sensitive AHAS, together with the malathion revisable resistance, suggested that herbicide metabolism likely plays a main role in the mechanism of mesosulfuron-methyl resistance in the SX population. To our knowledge, this is the first report elucidating the mesosulfuron-methyl resistance in Tausch's goatgrass.

Investigating the mechanisms of glyphosate resistance in goosegrass (Eleusine indica (L.) Gaertn.) by RNA sequencing technology.

Glyphosate is an important non-selective herbicide that is in common use worldwide. However, evolved glyphosate-resistant (GR) weeds significantly affect crop yields. Unfortunately, the mechanisms underlying resistance in GR weeds, such as goosegrass (Eleusine indica (L.) Gaertn.), an annual weed found worldwide, have not been fully elucidated. In this study, transcriptome analysis was conducted to further assess the potential mechanisms of glyphosate resistance in goosegrass. The RNA sequencing libraries generated 24 597 462 clean reads. De novo assembly analysis produced 48 852 UniGenes with an average length of 847 bp. All UniGenes were annotated using seven databases. Sixteen candidate differentially expressed genes selected by digital gene expression analysis were validated by quantitative real-time PCR (qRT-PCR). Among these UniGenes, the EPSPS and PFK genes were constitutively up-regulated in resistant (R) individuals and showed a higher copy number than that in susceptible (S) individuals. The expressions of four UniGenes relevant to photosynthesis were inhibited by glyphosate in S individuals, and this toxic response was confirmed by gas exchange analysis. Two UniGenes annotated as glutathione transferase (GST) were constitutively up-regulated in R individuals, and were induced by glyphosate both in R and S. In addition, the GST activities in R individuals were higher than in S. Our research confirmed that two UniGenes (PFK, EPSPS) were strongly associated with target resistance, and two GST-annotated UniGenes may play a role in metabolic glyphosate resistance in goosegrass.

Characterization of Eleusine indica with gene mutation or amplification in EPSPS to glyphosate.

The evolution of weed-resistant species threatens the sustainable use of glyphosate, which is the most important herbicide widely used in agriculture worldwide. Moreover, the high glyphosate resistance (>180-fold based on LD50) of Eleusine indica found in Malaysia, which carries a double mutation in its 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), made the control of this species more difficult. By contrast, the same species carrying the same double mutation in EPSPS (T102I+P106S) but found in China only shows a resistance level of not more than 14-fold based on GR50. The resistance level of this population is four times higher than that of the population carrying a single mutation (P106L). Although the members of this population survive under a high glyphosate dosage of 10,080gaeha-1, their growth was significantly inhibited by glyphosate under the recommend dose (840gaeha-1), where in the fresh weight was 85.4% of the control. EPSPS expression, relative copy number, and EPSPS activity in this population were similar to those of the susceptible population. In addition, the expression of two glutathione transferase (GST) genes (GST-U8 and GST-23) and the enzyme activity of the GST in this population did not significantly differ from those of the susceptible population. This finding is important in elucidating the resistance of the naturally evolved glyphosate-resistant (GR) weed species carrying a double mutation in EPSPS to glyphosate.

Target-site basis for resistance to imazethapyr in redroot amaranth (Amaranthus retroflexus L.).

Experiments were conducted to confirm imazethapyr resistance in redroot amaranth (Amaranthus retroflexus L.) and study the target-site based mechanism for the resistance. Whole-plant response experiments revealed that the resistant (R) population exhibited 19.16 fold resistance to imazethapyr compared with the susceptible (S) population. In vitro ALS activity assay demonstrated that the imazethapyr I50 value of the R population was 21.33 times greater than that of the S population. However, qRT-PCR analysis revealed that there is no difference in ALS gene expression between the R and S populations. Sequence analysis revealed an Asp-376-Glu substitution in ALS in the R population. In order to verify that the imazethapyr resistance was conferred by Asp-376-Glu mutation, the ALS-R and ALS-S genes were fused to the CaMV 35S promoter and introduced into Arabidopsis respectively. The expression of ALS-R in transgenic Arabidopsis plants exhibited 13.79 fold resistance to imazethapyr compared to ALS-S transgenic Arabidopsis.

Mutations and amplification of EPSPS gene confer resistance to glyphosate in goosegrass (Eleusine indica).

MAIN CONCLUSION: Field-evolved resistance of goosegrass to glyphosate is due to double or single mutation in EPSPS , or amplification of EPSPS leads to increased transcription and protein levels. Glyphosate has been used widely in the south of China. The high selection pressure from glyphosate use has led to the evolution of resistance to glyphosate in weeds. We investigated the molecular mechanisms of three recently discovered glyphosate-resistant Eleusine indica populations (R1, R2 and R3). The results showed that R1 and R2 had double Thr102Ile and Pro106Ser mutation and a single mutation of Pro106Leu in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, respectively. Escherichia coli containing the mutated EPSPS genes was tolerant to glyphosate. EPSPS activity in R1 and R2 plants was higher than in the sensitive plants. There was no amino acid substitution in EPSPS gene in R3. However, expression of EPSPS in R3 plants was higher than in glyphosate-susceptible (S) population (13.8-fold) after glyphosate treatment. EPSPS enzyme activity in both R3 and S plants was inhibited by glyphosate, while shikimate accumulation in R3 was significantly lower than for the S population. Further analysis revealed that the genome of R3 contained 28.3-fold more copies of the EPSPS gene than that of susceptible population. EPSPS expression was positively correlated with copy number of EPSPS. In conclusion, mutation of the EPSPS gene and increased EPSPS expression are part of the molecular mechanisms of resistance to glyphosate in Eleusine indica.

Molecular basis of resistance to imazethapyr in redroot pigweed (Amaranthus retroflexus L.) populations from China.

Three putative resistant Amaranthus retroflexus L. populations were collected in Heilongjiang province in China. Whole plant bioassays indicated high resistance (RI > 10) to imazethapyr in the three populations. In vitro acetolactate synthase (ALS) assays revealed that ALS from populations H3, H17 and H39 was less sensitive to imazethapyr inhibition compared to the susceptible population H76. The half-maximal inhibitory concentration (I50) values for H3, H17 and H39 were 14.83, 15.27 and 268 times greater, respectively, than that of the susceptible population H76. Three nucleotide mutations resulted in three known resistance-endowing amino acid substitutions, Ala-205-Val, Trp-574-Leu and Ser-653-Thr in the three resistant populations respectively. Therefore, ALS target-site mutations in resistant A. retroflexus could be responsible for imazethapyr resistance.

Molecular cloning and characterization of 5-enolpyruvylshikimate-3-phosphate synthase gene from Convolvulus arvensis L.

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS), the target enzyme for glyphosate inhibition, catalyzes an essential step in the shikimate pathway for aromatic amino acid biosynthesis. The full-length cDNA of 1,751 nucleotides (CaEPSPS, Genbank accession number: EU698030) from Convolvulus arvensis was cloned and characterized. The CaEPSPS encodes a polypeptide of 520 amino acids with a calculated molecular weight of 55.5 kDa and an isoelectric point of 7.05. The results of homology analysis revealed that CaEPSPS showed highly homologous with EPSPS proteins from other plant species. Tissue expression pattern analysis indicated that CaEPSPS was constitutively expressed in stems, leaves and roots, with lower expression in roots. CaEPSPS expression level could increase significantly with glyphosate treatment, and reached its maximum at 24 h after glyphosate application. We fused CaEPSPS to the CaMV 35S promoter and introduced the chimeric gene into Arabidopsis. The resultant expression of CaEPSPS in transgenic Arabidopsis plants exhibited enhanced tolerance to glyphosate in comparison with control.

Surgical treatment of neonatal Cantrell pentalogy: a case report and literature review.

Background: Pentalogy of Cantrell (PC) is a congenital multiple malformation consisting of midline supraumbilical thoracoabdominal wall defects, anterior and pericardial diaphragm defects, lower sternum defects, ectopia cordis and various intracardiac anomalies, that present a distinctive challenge for care-providers and surgeons. Case Description: Cases of PC in twin pregnancies is rare. We report a new born infant, weighing 2,400 g, the younger twin. After birth, he was transferred to the Children's Hospital of Soochow University due to the prenatal echocardiography indicated that he has ventricular septal defect (VSD), atrial septal defect (ASD) and aortic arch hypoplasia. From the appearance, the lower end of sternum is suspiciously missing, the apical beating point is located at the lower edge of the median xiphoid process of sternum, and the upper abdominal muscle below the beating area is defective. He has the clinical and imaging features of complete type of PC. Three surgical operations were performed in the neonatal period, including end-to-side anastomosis of aortic arch under cardiopulmonary bypass (CPB), patch repair of VSD, repair of ASD, ligation of ductus arteriosus, correction of cardiac vascular malformation and repair of chest and abdominal wall defect, and the postoperative recoveries went smoothly. Conclusions: PC is a rare congenital dysplasia, and its condition is complex. Our case shows that surgery is an effective treatment method, and the prognosis is related to the complexity of malformations.

Identification and characterization of pancreatic infections in severe and critical acute pancreatitis patients using 16S rRNA gene next generation sequencing.

Objectives: This study aimed to identify the bacterial composition in the pancreatic fluid of severe and critical acute pancreatitis (SAP and CAP) patients. Methods: A total of 78 pancreatic fluid samples were collected from 56 SAP and CAP patients and analyze using aerobic culture and 16S rRNA gene next-generation sequencing. The clinical data of the patients were obtained from the electronic medical records. Results: Among the total 78 samples, 16S rRNA gene NGS identified a total of 660 bacterial taxa, belonging to 216 species in 123 genera. The dominant aerobic bacteria included Klebsiella pneumoniae, Acinetobacter baumannii, and Enterococcus faecium, while the dominant anaerobic bacteria included Bacteroides, Dialister invisus, and Olsenella uli. As compared to aerobic culturing, 95.96% (95/99) of the aerobic cultured bacteria were detected using the 16S rRNA gene NGS. Conclusion: The pancreatic infections in SAP and CAP patients might originate not only from the gut but also from the oral cavity and airways as well as related environments. Dynamic analysis of bacterial profile and abundance showed that some bacteria with low abundance might become the main pathogenic bacteria. There were no significant differences in the bacterial diversity between SAP and CAP.