Purpose:To evaluate multiple biomarkers of colorectal tumor and their potential usage in automated diagnosis of colorectal cancer.Methods:10 normal colonic mucosae, 30 colorectal adenomas and 53 colorectal carcinomas with complete follow-up data were examined for multiple biomarkers(DNA contents, AgNOR, PCNA and nuclear morphometry) with quantitative computed processed imaging analysis.Results:The values of most of the parameters analyzed increased in accordance with the normal mucosa-adenoma-adenocarcinoma sequence, indicating a marked tendency for progression of colorectal malignancy. Among those parameters, DNA index,positive rates, counts, density and aspect factor of AgNOR, positive rates and density of PCNA, shape factor, width and density of nucleus were demonstrated to be relatively valuable indices.Conclusions:The quantitative evaluation of some indices of colorectal tumor can provide reproducible data for differential diagnostic of this tumor.

Objective To quantify the percentage of CD8+ T cells and their expressions of cytotoxic molecules and homing-related chemokine receptors in peripheral blood from patients with atopic dermatitis (AD).Methods Peripheral blood was obtained from 15 patients with AD and 14 healthy controls.Flow cytometric analysis was performed to determine the percentages of CD8+ T cells and CD8+CLA+ T cells in the peripheral blood samples,as well as the expression levels of cytotoxic molecules and homing-related chemokine receptors on these cells.Differences in these parameters were analyzed using t test,and relationship between these parameters was evaluated using Pearson correlation coefficient.Results No significant difference was observed between the patients with AD and healthy controls in the percentage of CD8+ T cells,the expressions of perforin,granzyme B,CCR10,CCR6 or FasL on CD8+ T cells,or the expressions of CCR4,CCR10,CXCR6 or FasL on CLA+CD8+ T cells (all P ＞ 0.05).A significant increase was noted in the percentage of CLA+CD8+ T cells (3.80％ ± 1.46％ vs.2.18％ ± 0.85％,t =3.636,P ＜ 0.01) and expression rates of CCR4 on CD8+ T cells (13.86％ ± 4.42％ vs.9.50％ ± 2.14％,t =3.738,P ＜ 0.01) as well as perforin and granzyme B on CLA+CD8+ T cells (74.27％ ± 15.94％ vs.57.20％ ± 14.64％,t =2.998,P ＜ 0.01; 70.90％ ± 13.85％ vs.56.41％ ± 11.00％,t =3.104,P ＜ 0.01) in the patients with AD compared with the healthy controls.Conclusions The proportion of CLA+CD8+ T cells is increased with enhanced expressions of cytotoxic molecules such as perforin and granzyme B in peripheral blood of patients with AD,which may contribute to the pathogenesis of AD.

Objective: To investigate the effects of andrographolide on extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and tumor necrosis factor-α (TNF-α) expression in lipopolysaccharide (LPS)-activated macrophages. Methods: LPS-activated mouse peritoneal macrophages were cultured in media with different concentrations of andrographolide. Cytotoxicity of andrographolide was detected by cell counting kit-8. The macrophages were lysed, and then expressions of phosphorylated ERK1/2, JNK and p38 and nuclear factor-κB inhibitor (IκBα) protein were detected by Western blotting and TNF-α mRNA expression was detected by reverse transcription-polymerase chain reaction. Supernatants of the macrophages were used to detect content of TNF-α protein by enzyme-linked immunosorbent assay. Results: Andrographolide at 1-100 μg/mL showed no cytotoxicity on LPS-activated mouse peritoneal macrophages. Andrographolide inhibited ERK1/2 phosphorylation in LPS-activated murine peritoneal macrophages, which was concentration-dependent (P<0.01). Andrographolide at 1-25 μg/mL had no effects on phosphorylation levels of JNK and p38 and IκBα degradation in LPS-stimulated mouse peritoneal macrophages. In activated macrophages, TNF-α expression was inhibited by 12 μg/mL andrographolide and 20 μmol/L PD98059 (inhibitor of ERK1/2 signaling pathway) at both mRNA expression and protein secretion levels. Conclusion: In LPS-activated macrophages, andrographolide may inhibit the expression of TNF-α by inhibiting ERK1/2 signaling pathway.

Hepatolenticular Degeneration ; blood ; drug therapy ; Humans ; Sodium ; therapeutic use ; Suburethral Slings

Objective: To investigate the clinical effect of free medical sural artery perforator myocutaneous flap for repairingof the woundwith osteomyelitis. Methods: 17 patients suffered from the wound with osteomyelitis were treated in the Ningbo First People′s Hospital, There were 11 males and 6 females with an mean age of 53.2 years (range, 21-76 years). The sizes of the defect ranged 5 cm×4 cm to 13 cm×6 cm. All patients underwent debridement and used antibiotic-loaded bonecement to cover the wound. Meanwhile, patients were treated with sensitive antibiotics, operation and free medical sural artery perforator myocutaneous flap were used to treat the wound. Preoperative use ultrasound and CT angiography to positioning perforator, The flap area ranged from 6 cm×4 cm to 13 cm×7 cm and the donor sites were closed directly. The author provided the patients with the treatment of anti-inflammatory, anti-spasmodic and anti-coagulantin the postoperative. Used infrared thermograms to assess the flap blood supply. Results: One flap skin margin was non union due to poor blood supply.All of the other 16 flaps success survived and the donor sites were closed directly. Postoperative follow-up period was 4 to 23 months and the flaps had satisfied texture and appearance.All the donor sites had a good healing with no pain and complications, also the osteomyelitis was controlled. Conclusions The free medical sural artery perforator myocutaneous flap is reliable for reconstruction of the wound with osteomyelitis.

Objective To study the effect of hepatitis E virus Open Reading Frame 3 protein on the proliferation and cell cycle of Chang liver cells (CCL13).Methods The pDsRed-Monomer-N1-ORF3 recombinant plasmid was transfected into CCL13 cells via lipofectamine 2 000 reagent,then assayed cell proliferation by MTT assay;Flow cytometric was used to detect cell cycle after dying with PI;and the cell cycle proteins expression were detected by western-blotting.Results pDsRed-Monomer-N1-ORF3 recombinant plasmid was successfully transfected into CCL13 cells,Expression of ORF3 can inhibit the proliferation activity of CCL13 cells from the 3rd day (P ＜ 0.01),and prevent the cell cycle in G0/G1 phase;Western Blot showed expression of cell cycle proteins cyclin D1 and cyclin E1 were decreased in ORF3 group compared with the control group;However,the expression of cyclin A2 and cyclin B1 had no significantly difference in three groups.Conclusions HEV-ORF3 can inhibits the proliferation activity of CCL13 cells by inhibition expression of cell cycle proteins cyclin D1 and cyclin E1.

To investigate the clinical efficacy of one-stage repairing the wounds after excision of soft tissue malignant tumor in lower limb with deep inferior epigastric perforator (DIEP) flaps. Methods From Au-gust, 2016 to July, 2018, 11 patients(8 males and 3 females; aged 35-76 years) with the soft tissue defects in the low-er limb underwent immediate reconstructive operations with DIEP flap after their tumor resection. The defects ranged in area from 11 cm×7 cm to 25 cm×10 cm after extended resection, including 5 cases of bone exposure. The DIEP flap area ranged from 11 cm×8 cm to 26 cm×18 cm.Among them, 9 cases were treated with free transplantation and 2 cases with pedicle transplantation.The donor sites were closed directly. The patients’ limb function, tumor recurrence and the healing of surgical incision had been followed-up. Results Eleven flaps survived and the donor sites healed directly. One flap had skin pigmentation due to postoperative vascular crisis, and the other flaps had satisfied texture and appearance. All patients were followed-up from 6 to 30 months. The lower limb movement was normal. All the donor sites had a good healing with no pain and abdominal complications. No tumor recurrence was observed during the followed-up period. Conclusion DIEP flap have many advantages, such as large cutting area, which is suitable for large-area wound repair after extended excision of soft tissue malignant tumor in lower limb. At the same time, the perforator vessel is constant and the caliber are thick, also the donor area is concealed and can be sutured directly.This kind of the technique for reconstruction of lower limb soft tissue defects after malignant tumor resection is well acceptable.

Objective:To explore the feasibility and clinical effectiveness of preoperative positioning grid combined with CTA assisted design of anterolateral thigh perforator flap (ALTPF) in reconstruction of soft tissue defects around foot and ankle.Methods:From May 2018 to December 2021, a total of 18 cases with soft tissue defects around foot and ankle were admitted to the Department of Plastic and Reconstructive Surgery, First Affiliated Hospital of Ningbo University. The patients were 11 males and 7 females, aged from 32 to 78 years old, with an average age of 57.5 years old. Among them, 10 had chronic ulcer wounds, 6 had traumatic wounds, and 2 had postoperative wounds after malignant tumor resection. The sizes of soft tissue defect ranged from 6.0 cm×8.0 cm to 9.0 cm×13.0 cm, and the flap sizes ranged from 8.0 cm×10.0 cm to 11.0 cm×15.0 cm. Preoperative positioning grid combined with CTA three-dimensional reconstruction were used to mark the exit point of the perforator vessels in digital format, in order to restore the course of vessels and calculate the length of the vascular pedicles. ALTPFs were accurately designed based on the digitally reconstructed images, and then the ALTPFs were used to repair the soft tissue defects around the ankle. The flap donor sites were directly closed in stage Ⅰ. After the operation, all the patients were included in scheduled follow-ups at the outpatient department to observe the appearance of the recipient flaps and donor sites. The functional evaluation of the affected feet were assessed according to the American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot score.Results:All 18 flaps survived, and the wounds in both the recipient and donor sites healed in stageⅠ. Postoperative follow-up for the 18 patients were 5 to 36 months, with an average of 13 months. At the last follow-up, the appearances of the flaps were good without swelling, the donor sites had good recovery, and there was no obvious scar hypertrophy. The function evaluation of the affected feet were found at excellent in 10 cases, good in 6 cases, and fair in 2 cases.Conclusion:Preoperative position grid combined with CTA three-dimensional reconstruction can digitally mark the exit points and running courses of the perforator vessels. It is an effective method for accurate position of perforator vessels before surgery. It can effectively reduce the operation time, lower the surgical risks, and achieve a high survival rate of the flap, thus holding considerable clinical value.

Objective To evaluate the relationship between diabetic nephropathy(DN)and diabetic retinopathy(DR)in patients with type 2 diabetes mellitus(T2DM)based on imaging and clinical testing data.Methods Totally 600 T2DM patients who visited the First People's Hospital of Ziyang from March 2021 to December 2022 were included.The fundus photography and fundus fluorescein angiography were performed on all these patients and their age,gender,T2DM duration,cardiovascular diseases,cerebrovascular disease,hypertension,smoking history,drinking history,body mass in-dex,systolic blood pressure,diastolic blood pressure and other clinical data were collected.The levels of fasting blood glu-cose(FPG),triglyceride(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),low-density lipo-protein cholesterol(LDL-C),glycosylated hemoglobin(HbA1c),24 h urinary albumin(UAlb),urinary albumin to creati-nine ratio(ACR),serum creatinine(Scr)and blood urea nitrogen(BUN)were measured.Logistic regression was used to analyze the risk factors associated with DR.DR staging was performed according to fundus images,and the convolutional neural network(CNN)algorithm was used as an image analysis method to explore the correlation between DR and DN based on emission computed tomography(ECT)and clinical testing data.Results The average lesion area rates of DR and DN detected by the CNN in the non-DR,mild-non-proliferative DR(NPDR),moderate-NPDR,severe-NPDR and pro-liferative DR(PDR)groups were higher than those obtained by the traditional algorithm(TCM).As DR worsened,the Scr,BUN,24 h UAlb and ACR gradually increased.Besides,the incidence of DN in the non-DR,mild-NPDR,moderate-NPDR,severe-NPDR and PDR groups was 1.67％,8.83％,16.16％,22.16％and 30.83％,respectively.Logistic regression analysis showed that the duration of T2DM,smoking history,HbA1c,TC,TG,HDL-C,LDL-C,24 h UAlb,Scr,BUN,ACR and glomerular filtration rate(GFR)were independent risk factors for DR.Renal dynamic ECT analysis demonstrated that with the aggravation of DR,renal blood flow perfusion gradually decreased,resulting in diminished renal filtration.Conclusion The application of CCN in the early stage DR and DN image analysis of T2DM patients will improve the diag-nosis accuracy of DR and DN lesion area.The DN is worsening as the aggravation of DR.

Alfalfa(Medicago sativa L.)is the most important legume forage crop worldwide with high nutritional value and yield.For a long time,the breeding of alfalfa was hampered by lacking reliable information on the autotetraploid genome and molecular markers linked to important agro-nomic traits.We herein reported the de novo assembly of the allele-aware chromosome-level genome of Zhongmu-4,a cultivar widely cultivated in China,and a comprehensive database of genomic variations based on resequencing of 220 germplasms.Approximate 2.74 Gb contigs(N50 of 2.06 Mb),accounting for 88.39％of the estimated genome,were assembled,and 2.56 Gb contigs were anchored to 32 pseudo-chromosomes.A total of 34,922 allelic genes were identified from the allele-aware genome.We observed the expansion of gene families,especially those related to the nitrogen metabolism,and the increase of repetitive elements including transposable elements,which probably resulted in the increase of Zhongmu-4 genome compared with Medicago truncatula.Population structure analysis revealed that the accessions from Asia and South America had rela-tively lower genetic diversity than those from Europe,suggesting that geography may influence alfalfa genetic divergence during local adaption.Genome-wide association studies identified 101 sin-gle nucleotide polymorphisms(SNPs)associated with 27 agronomic traits.Two candidate genes were predicted to be correlated with fall dormancy and salt response.We believe that the allele-aware chromosome-level genome sequence of Zhongmu-4 combined with the resequencing data of the diverse alfalfa germplasms will facilitate genetic research and genomics-assisted breeding in variety improvement of alfalfa.