Genomic and transcriptomic analysis unveils population evolution and development of pesticide resistance in fall armyworm Spodoptera frugiperda

The fall armyworm (FAW), Spodoptera frugiperda, is a destructive pest native to America and has recently become an invasive insect pest in China. Because of its rapid spread and great risks in China, understanding of FAW genetic background and pesticide resistance is urgent and essential to develop effective management strategies. Here, we assembled a chromosome-level genome of a male FAW (SFynMstLFR) and compared re-sequencing results of the populations from America, Africa, and China. Strain identification of 163 individuals collected from America, Africa and China showed that both C and R strains were found in the American populations, while only C strain was found in the Chinese and African populations. Moreover, population genomics analysis showed that populations from Africa and China have close relationship with significantly genetic differentiation from American populations. Taken together, FAWs invaded into China were most likely originated from Africa. Comparative genomics analysis displayed that the cytochrome p450 gene family is extremely expanded to 425 members in FAW, of which 283 genes are specific to FAW. Treatments of Chinese populations with twenty-three pesticides showed the variant patterns of transcriptome profiles, and several detoxification genes such as AOX, UGT and GST specially responded to the pesticides. These findings will be useful in developing effective strategies for management of FAW in China and other invaded areas.

Correlation between angiotensin H level and clinical inflammatory indicators in patients with rheumatoid arthritis and therapeutic effect of angiotensin receptor blockers / 中国药理学通报

Aim To investigate the eorrelation between angiotensin II (Ang II ) level and clinical indicators in patients with rheumatoid arthritis ( HA) , and to determine the therapeutic effect of angiotensin receptor blockers ( ARBs).Methods Plasma samples and personal information were collected from HA patients admitted to our hospital from 2019 to 2021.The level of Ang II in plasma was determined by ELISA to elucidate the correlation between plasma Ang II level and the severity of HA.The pathological changes of synovi-al tissues and T eells subtype in different groups of HA patients were determined by pathological examination and flow cytometry.A rat model of collagen-induced arthritis (CIA) was established and the pathological examination was used to confirm that valsartan could alleviate the disease course in the CIA animal model.Results Compared with control group, the plasma level of Ang II in HA patients significantly increased.After therapy with oral ARBs plasma Ang H levels and anti - cyclic citrullinated peptide antibody ( CCP) titre were significantly lower than those untreated HA patients.The level of Ang II in plasma was positively correlated with CCP and the number of monocytes, but negatively with number of RBC and hemoglobin content.Staining of synovial tissue with HE and Masson found that patients with HA had significant synovial proliferation, pannus formation , and numerous inflammatory cell infiltrates compared with control patients.Immunohistochemical results showed significant infiltration of CD4 4 T cells in synovial tissues of HA patients.Western blot and immunofluorescence analysis showed that the expression of angiotensin type 1 receptor ( ATI R ) was significantly up-regulated in CD4 + T cells and synovial tissues of HA patients.The results of animal experiments showed that valsartan harl therapeutic effect on CIA rats and could delay the disease process of CIA.Conclusions Plasma Ang II level is positively correlated with CCP level and HA severity.ARBs can down-regualte CCP level and delay disease progression in HA patients.Animal experiments showed that valsartan blocks the combination of Ang H and ATI R and has therapeutic effect on a CIA rat model.This study provides the theoretical and experimental basis for ARBs to become the preferred antihypertensive drugs for HA patients with hypertension.

Lithium chloride prevents glucocorticoid-induced osteonecrosis of femoral heads and strengthens mesenchymal stem cell activity in rats / 中华医学杂志(英文版)

BACKGROUND@#Accumulating evidence suggests that lithium influences mesenchymal stem cell (MSC) proliferation and osteogenic differentiation. As decreased bone formation in femoral heads is induced by glucocorticoids (GCs), we hypothesized that lithium has a protective effect on GC-induced osteonecrosis of femoral heads (ONFH).@*METHODS@#A rat ONFH model was induced by methylprednisolone (MP) and the effect of lithium chloride on the models was evaluated. Micro-computed tomography (CT)-based angiography and bone scanning were performed to analyze the vessels and bone structure in the femoral heads. Hematoxylin and eosin and immunohistochemical staining were performed to evaluate the trabecular structure and osteocalcin (OCN) expression, respectively. Bone marrow-derived MSCs were isolated from the models, and their proliferative and osteogenic ability was evaluated. Western blotting and quantitative real-time polymerase chain reaction were performed to detect osteogenic-related proteins including Runx2, alkaline phosphatase, and Collagen I.@*RESULTS@#Micro-CT analysis showed a high degree of osteonecrotic changes in the rats that received only MP injection. Treatment with lithium reduced this significantly in rats that received lithium (MP + Li group); while 18/20 of the femoral heads in the MP showed severe osteonecrosis, only 5/20 in the MP + Li showed mild osteonecrotic changes. The MP + Li group also displayed a higher vessel volume than the MP group (0.2193 mm3vs. 0.0811 mm3, P < 0.05), shown by micro-CT-based angiography. Furthermore, histological analysis showed better trabecular structures and more OCN expression in the femoral heads of the MP + Li group compared with the MP group. The ex vivo investigation indicated higher proliferative and osteogenic ability and upregulated osteogenic-related proteins in MSCs extracted from rats in the MP + Li group than that in the MP group.@*CONCLUSIONS@#We concluded that lithium chloride has a significant protective effect on GC-induced ONFH in rats and that lithium also enhances MSC proliferation and osteogenic differentiation in rats after GC administration.

Intrauterine balloon tamponade combined with temporary abdominal aortic balloon occlusion in the management of women with placenta accreta spectrum:a randomized controlled trial / 中华妇产科杂志

Objective:To access the effectiveness and safety of the intrauterine balloon tamponade verse gauze packing combined with temporary abdominal aortic balloon occlusion in the management of placenta accreta spectrum (PAS).Methods:This was an open-label, randomized controlled trial conducted in Nanjing Drum Tower Hospital. The patients suspected with PAS for uterine preservation surgery under the multidisciplinary team care were recruited between Aug 2015 and Jan 2018. When bleeding could not be achieved after fetus delivered, and a temporary abdominal aortic balloon occlusion and the compression sutures as needed, the women were randomly allocated 1∶1 into balloon tamponade ( n=81) or gauze packing ( n=80) group. The primary outcome was successful bleeding arrests by avoiding second line surgeries. The secondary outcomes included the volume of blood loss during and after cesarean section, the rate of PPH, incidence and amount of blood transfusion, hysterectomy, postpartum pain, ICU admission, need for re-laparotomy, and the length of hospital stay, readmission, and interventional radiology complications. Results:All the women [100% (81/81)] in the balloon group were obtained hemostasis without further intervention, significantly higher than 88% (70/80) in the gauze group ( P=0.001). Before uterine tamponade, blood loss were 820 ml (620-1 230) ml and 850 ml (605-1 442) ml, while placenta bed were sutured in 96%（78/81, 77/80） respectively ( P>0.05).The proportion of blood loss≥1 000 ml was higher in the gauze group than that in the balloon group ( P=0.006). Maternal adverse events involving total blood loss, puerperal morbidity and postpartum pain occurred more frequently in the gauze group ( P<0.05). The following outcome showed no statistically significant difference between the two groups: the vascular occlusion time, the dose of radiation, and interventional radiology complication ( P>0.05). The median volume infused into the lower and upper balloons is 70 ml (50-100 ml) and 180 ml (100-240 ml). Conclusions:Incrauterine balloon tamponade is as effective as gauze packing in hemostasis following the placenta delivery in PAS. Compared with gauze packing, the uterine balloon tamponade is more effective.

Quality Evaluation of Physical Properties of Ultrafine Granular Powder of Salviae Miltiorrhizae Radix et Rhizoma / 中国实验方剂学杂志

Objective:Taking ultrafine granular powder of Salviae Miltiorrhizae Radix et Rhizoma （UGPSMR） as the research object， to establish a method for evaluating its physical properties. Method:A method was established for measuring the particle size distribution and specific surface area of UGPSMR， and the methodological investigation was carried out. A total of 15 physical indicators ［D90 （particle size value when the cumulative particle distribution reaches 90%）， particle size distribution range， particle size distribution width， bulk density， tap density， intergranular porosity， Carr index， specific surface area， pore volume， angle of repose， tablet angle， Hausner ratio， black to white degree L*， red to green degree a*， yellow to blue degree b*］ were used to characterize the quality attributes of UGPSMR and to construct the physical fingerprint. Multivariate statistical analysis methods such as similarity analysis， cluster analysis （CA）， principal component analysis （PCA） and orthogonal partial least squares discriminant analysis （OPLS-DA） were used to evaluate the quality of 11 batches of UGPSMR （S1-S11） produced in different years， and to find out the difference index between samples from different batches. Result:The method for measuring the particle size distribution and specific surface area of UGPSMR was feasible and repeatable. The similarities between the physical fingerprint of 10 batches of samples （S1-S3， S5-S11） from production and control fingerprint of UGPSMR were above 0.85， but the similarity between sample S4 and the control fingerprint was only 0.488. There were some differences in physical property indicators between different batches of UGPSMR， and the characteristic difference indicators were intergranular porosity， specific surface area， pore volume， b*， L*， Carr index， particle size distribution width， respectively. Conclusion:This method can comprehensively evaluate the physical quality attributes of UGPSMR， and can reflect the effect of differences in material basis of the medicinal materials or production process on the physical properties of finished products， and can evaluate quality consistency between batches from the physical state level， which provides new ideas for the quality control of ultrafine granular powder of herbal medicine.

Quality evaluation of angelica broken wall powder based on QbD concept of traditional Chinese medicine / 中国中药杂志

In this paper, angelica broken wall powder(ABWP) was taken as the research object, HPLC fingerprint combined with multi-component determination(ferulic acid, senkyunolide I, coniferyl ferulate, ligustilide and 3-butylidenephthalide), physical fingerprint(D_(90), particle size distribution range, particle size distribution width, bulk density, tap density, inter-particle porosity, Carr index, specific surface area, pore volume, angle of repose, Hausner ratio, loss on drying and hygroscopicity)were used to characterize the quality attribute of ABWP; similarity analysis, cluster analysis, principal component analysis and orthogonal partial least squares discriminant analysis were conducted to construct the quality evaluation method of holographic analysis based on traditional Chinese medicine QbD "4 H mode", in order to evaluate the quality of ABWP from different sources and find out differentiated indicators. The quality evaluation method could be used for scientific, comprehensive evaluation of the quality attribute of ABWP, and the quality consistency evaluation of cell-wall-broken powder of different sources or different processes.It provides new ideas for quality control and research of ultrafine granular powders of traditional Chinese medicine.

Lactobacillus rhamnosus GG conditioned medium prevents E. coli meningitis by inhibiting nuclear factor-κB pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate whether Lactobacillus rhamnosus GG conditioned medium(LGG-CM)has preventive effect against E. coli K1-induced neuropathogenicity in vitro by inhibiting nuclear factor-κB (NF-κB) signaling pathway.</p><p><b>METHODS</b>An in vitro blood-brain barrier (BBB) model was constructed using human brain microvascular endothelial cells (HBMECs). The effect of LGG-CM on E. coli-actived NF-κB signaling pathway was assayed using Western blotting. Invasion assay and polymorphonuclear leukocyte (PMN) transmigration assay were performed to explore whether LGG-CM could inhibit E. coli invasion and PMN transmigration across the BBB in vitro. The expressions of ZO-1 and CD44 were detected using Western blotting and immunofluorescence. The changes of trans-epithelial electric resistance (TEER) and bacterial translocation were determined to evaluate the BBB permeability.</p><p><b>RESULTS</b>Pre-treament with LGG-CM inhibited E. coli-activated NF-κB signaling pathway in HBMECs and decreased the invasion of E. coli K1 and transmigration of PMN. Western blotting showed that LGG-CM could alleviate E. coli-induced up-regulation of CD44 and down-regulation of ZO-1 expressions in HBMECs. In addition, pre-treatment with LGG-CM alleviated E. coli K1-induced reduction of TEER and suppressed bacterial translocation across the BBB in vitro.</p><p><b>CONCLUSION</b>LGG-CM can block E. coli-induced activation of NF-κB signaling pathway and thereby prevents E. coli K1-induced neuropathogenicity by decreasing E. coli K1 invasion rates and PMN transmigration.</p>

Dissolution difference of ginsenosides from ultrafine granular powder and common powder traditional pieces of Panacis Quinquefolii Radix in vitro / 中国中药杂志

The dissolution of Panacis Quinquefolii Radix ultrafine granular powder and common powder, traditional pieces in water and simulated gastric juice in vitro was compared, and the effect of particles size of Panacis Quinquefolii Radix on the dissolution was studied. HPLC method was used for determination of five ginsenosides including Rg1, Re, Rb1, Rc and Rd from ultrafine granular powder and common powder, traditional pieces of Panacis Quinquefolii Radix at different points in time, furthermore, the dissolution curves of Panacis Quinquefolii Radix ultrafine granular powder and common powder, traditional pieces were obtained. The dissolution characteristics of the three Panacis Quinquefolii Radix forms were also compared in this study. According to the results, the dissolution rates of ginsenosides from ultrafine granular powder exceeded 90% of the total content with 5 min, significantly higher than that of the other two forms in water in vitro. At the same time, the dissolved amount of the ultrafine granular powder was fourteen percent higher than that of the traditional pieces and eight percent higher than that of the common powder. Under the condition of simulated gastric juice in vitro, the dissolution rates of ginsenosides from ultrafine granular powder were little lower than that of the other two, but the maximum dissolved amount of the former was fourteen percent higher than that of the common powder and five percent higher than that of the extracts. Therefore the conclusion is that micronization of Panacis Quinquefolii Radix contributed to dissolution of effective components.

Preliminary study on effect of Rhodiolae Crenulatae Radix et Rhizoma cell wall-broken decoction pieces on intestinal flora of mice / 中国中药杂志

This study aims to analyze and compare the effect of cell wall-broken decoction pieces, conventional decoction pieces and conventional powder of Rhodiolae Crenulatae Radix et Rhizoma on the intestinal flora of normal mice. The conventional bacterial culture and PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) were adopted for the mice after the oral administration for 14 days. According to the bacterial culture results, the 1/8 dose cell wall-broken decoction pieces group showed fewer Enterococcus and Escherichia coli bacillus but more Lactobacillus and Bifidobacterium than the conventional decoction pieces group and the traditional powder group (P <0.05). Meanwhile, on the basis of the PCR-DGGE results, the 1/8 dose cell wall-broken decoction pieces group revealed the highest Shannon-Wiener index (H) and species richness (S) among the seven groups, with extremely significant differences compared with the normal group (P <0.01), significant differences compared with the conventional decoction pieces group and the conventional powder group (P <0.05) and a high intra-group similarity. In conclusion, the long-term intake of 1/8 dose Rhodiolae Crenulatae Radix et Rhizoma cell wall-broken decoction pieces showed a certain effect in regulating intestinal tract by promoting the growth of Lactobacillus and Bifidobacterium. Furthermore, the intestinal flora community will become more stable.

Management of Benign Tracheal Stenosis by Small-diameter Tube-assisted Bronchoscopic Balloon Dilatation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>A limitation of bronchoscopic balloon dilatation (BBD) is that airflow must be completely blocked for as long as possible during the operation. However, the patient often cannot hold his or her breath for a long period affecting the efficacy of the procedure. In this study, we used an extra-small-diameter tube to provide assisted ventilation to patients undergoing BBD and assessed the efficacy and safety of this technique.</p><p><b>METHODS</b>Bronchoscopic balloon dilatation was performed in 26 patients with benign tracheal stenosis using an extra-small-diameter tube. The tracheal diameter, dyspnea index, blood gas analysis results, and complications were evaluated before and after BBD. Statistical analyses were performed by SPSS version 16.0 for Windows (SPSS, Inc., Chicago, IL, USA).</p><p><b>RESULTS</b>Sixty-three BBD procedures were performed in 26 patients. Dyspnea immediately improved in all patients after BBD. The tracheal diameter significantly increased from 5.5 ± 1.5 mm to 13.0 ± 1.3 mm (P < 0.001), and the dyspnea index significantly decreased from 3.4 ± 0.8 to 0.5 ± 0.6 (P < 0.001). There was no significant change in the partial pressure of oxygen during the operation (before, 102.5 ± 27.5 mmHg; during, 96.9 ± 30.4 mmHg; and after, 97.2 ± 21.5 mmHg; P = 0.364), but there was slight temporary retention of carbon dioxide during the operation (before, 43.5 ± 4.2 mmHg; during, 49.4 ± 6.8 mmHg; and after, 40.1 ± 3.9 mmHg; P < 0.001).</p><p><b>CONCLUSION</b>Small-diameter tube-assisted BBD is an effective and safe method for the management of benign tracheal stenosis.</p>

The species traceability of the ultrafine powder and the cell wall-broken powder of herbal medicine based on DNA barcoding / 药学学报

Ultrafine powder and cell wall-broken powder of herbal medicine lack of the morphological characters and microscopic identification features. This makes it hard to identify herb's authenticity with traditional methods. We tested ITS2 sequence as DNA barcode in identification of herbal medicine in ultrafine powder and cell wall-broken powder in this study. We extracted genomic DNAs of 93 samples of 31 representative herbal medicines (28 species), which include whole plant, roots and bulbs, stems, leaves, flowers, fruits and seeds. The ITS2 sequences were amplified and sequenced bidirectionally. The ITS2 sequences were identified using Basic Local Alignment Search Tool (BLAST) method in the GenBank database and DNA barcoding system to identify the herbal medicine. The genetic distance was analyzed using the Kimura 2-parameter (K2P) model and the Neighbor-joining (NJ) phylogenetic tree was constructed using MEGA 6.0. The results showed that DNA can be extracted successfully from 93 samples and high quality ITS2 sequences can be amplified. All 31 herbal medicines can get correct identification via BLAST method. The ITS2 sequences of raw material medicines, ultrafine powder and cell wall-broken powder have same sequence in 26 herbal medicines, while the ITS2 sequences in other 5 herbal medicines exhibited variation. The maximum intraspecific genetic-distances of each species were all less than the minimum interspecific genetic distances. ITS2 sequences of each species are all converged to their standard DNA barcodes using NJ method. Therefore, using ITS2 barcode can accurately and effectively distinguish ultrafine powder and cell wall-broken powder of herbal medicine. It provides a new molecular method to identify ultrafine powder and cell wall-broken powder of herbal medicine in the quality control and market supervision.

Based on the low-density cDNA Macroarray for screening of antiviral proteins of IFNa tissues / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To screen the gene expression profiles of IFN-alpha antiviral proteins based on a low-density cDNA Macroarray, and to explore the relationship between the expression of antiviral protein and the HBV replication.</p><p><b>METHODS</b>The HepG2 and HepG2.2.15 cells were treated with various concentrations of IFN-alpha (0 IU/ml, 100 IU/ml, 1000 IU/ml) of IFN-alpha for 6 h, and then the low-density cDNA Macroarray was used for analysing the expression profiles of antiviral genes and screening differential expressions of antiviral proteins. Meanwhile, the HepG2 cells were transiently transfected with HBV core protein-expressed plasmid pHBc-EGFP, and the expressions of antiviral proteins were analysed by RT-PCR assay. Moreover, the HepG2.2.15 cells were also transfected with the antiviral protein-expressed plasmid pcDNA3.1-Flag-MxA. ELISA was used for analysing the secreted HBV antigens, while dot blot and Southern blot were applied for analysing the extracellular HBV DNA and intracellular replicative intermediate HBV DNA in HepG2.2.15 cells. All data were presented as mean+/-SD and analyzed using the t-test and one-way analysis of variance (ANOVA) in the experiments.</p><p><b>RESULTS</b>The Macroarray results suggested that the expression of IFN-alpha antiviral genes like 6-16, IFITM1, IFITM2, IFITM3 and RING4 in HepG2.2.15 cells were partially inhibited. More importantly, it was found, in this research, the expression of antiviral protein MxA in HepG2.2.15 cells was completely suppressed. RT-PCR analysis indicated that the expression of MxA was also significantly decreased in HepG2 cells transfected with pHBc-EGFP plasmid. Although HepG2.2.15 cells transfected with pcDNA3.1-Flag-MxA plasmid could not inhibit extracellular HBV DNA and intracellular replicative intermediate HBV DNA, the MxA exerted some antiviral activities as it effectively suppressed the secretion of HBsAg and HBeAg in HepG2.2.15 cells.</p><p><b>CONCLUSIONS</b>HBV and its antigen components probably influence the expression of antiviral proteins. IFN- resistance may be related to the down-regulation of antiviral proteins expression.</p>

Study on NO(2) absorption by ascorbic acid and various chemicals / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Study on NO(2) absorption aimed at seeking a better NO(2) absorption chemical at pH 4.5 approximately 7.0 for application to existing wet flue gas desulfurization (FGD). The results from the double-stirred reactor indicated that ascorbic acid has very high absorption rate at this pH range. The rate constant of ascorbic acid reaction with NO(2) (0 approximately 1,000 x 10(-6) mol/mol) is about 3.54 x 10(6) mol/(Ls) at pH 5.4 approximately 6.5 at 55 degrees C.

Experimental study on the inhibition of biological reduction of Fe(III)EDTA in NO(x) absorption solution / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Scrubbing of NO(x) from the gas phase with Fe(II)EDTA has been shown to be highly effective. A new biological method can be used to convert NO to N(2) and regenerate the chelating agent Fe(II)EDTA for continuous NO absorption. The core of this biological regeneration is how to effectively simultaneous reduce Fe(III)EDTA and Fe(II)EDTA-NO, two mainly products in the ferrous chelate absorption solution. The biological reduction rate of Fe(III)EDTA plays a main role for the NO(x) removal efficiency. In this paper, a bacterial strain identified as Klebsiella Trevisan sp. was used to demonstrate an inhibition of Fe(III)EDTA reduction in the presence of Fe(II)EDTA-NO. The competitive inhibition experiments indicted that Fe(II)EDTA-NO inhibited not only the growth rate of the iron-reduction bacterial strain but also the Fe(III)EDTA reduction rate. Cell growth rate and Fe(III)EDTA reduction rate decreased with increasing Fe(II)EDTA-NO concentration in the solution.

Construction of the expression vectors of HDV ribozymes and their intracellular inhibiting activity against HCV RNA / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate whether HDV ribozymes can intracellularly inhibit HCV RNA.</p><p><b>METHODS</b>The mammalian expression vectors, pC1-RzC1, pC1-RzC2 and pC1-RzC3, containing ribozymes cDNA of RzC1, RzC2, and RzC3, were constructed targeting different HCV-5' NCR-C RNA regions. Then the HCV-positive fetal hepatocytes were transfected with these plasmids using liposome-mediated method. The inhibitory effects of HDV ribozymes were evaluated by HCV RNA quantitation in cultured cells and the supernatants.</p><p><b>RESULTS</b>(1) All the three HDV ribozymes were inserted into the expression vector. (2) Fetal hepatocytes were infected with HCV proven by RT-PCR and fluorescent quantitative PCR and expressed HCV NS3 and NS5 antigens by immunocytochemistry. (3) HDV ribozymes inhibited the activity of the target HCV RNA at expect positions in HCV-positive hepatocytes. At 0.5 micromol/L, the inhibitory rate of pC1-RzC1, pC1-RzC2, and pC1-RzC3 was 53.2%, 50.5 %, and 10.6% respectively. PC1-RzC1 was used continuously for one week, showing the inhibitory rate of 60.7%, 64.2%, 68.4%, 71.9%, 78.8% and 83.1% on the 2nd, 3rd, 4th, 5th, 6th and 7th day.</p><p><b>CONCLUSION</b>The inhibitory activity of pC1-RzC1 (107-113nt) and pC1-RzC2 (268-274nt) is greater than that of pC1-RzC3 (345-351nt) in HCV-positive hepatocytes.</p>

Inferior epigastric artery angiography applied in the transplantation with the deep inferior epigastric perforator free flap / 中华放射学杂志

Objective To observe the effect of inferior epigastric artery angiography applied in the transplantation with the deep inferior epigastric perforator free flap.Methods Seven patients who had undergone the deep inferior epigastric perforator free flap transplantation,received angiography of the inferior epigastric artery.The value of the angiography was discussed.Results All patients were successful in angiography without any adverse reaction.Mll patients were successful in transplantation except one because of personal reason.Conclusion Inferior epigastric artery angiography facilitates the transplantation with the deep inferior epigastrie perforator free flap.