Yeast cell detection using fuzzy automatic contrast enhancement (FACE) and you only look once (YOLO).

In contemporary biomedical research, the accurate automatic detection of cells within intricate microscopic imagery stands as a cornerstone for scientific advancement. Leveraging state-of-the-art deep learning techniques, this study introduces a novel amalgamation of Fuzzy Automatic Contrast Enhancement (FACE) and the You Only Look Once (YOLO) framework to address this critical challenge of automatic cell detection. Yeast cells, representing a vital component of the fungi family, hold profound significance in elucidating the intricacies of eukaryotic cells and human biology. The proposed methodology introduces a paradigm shift in cell detection by optimizing image contrast through optimal fuzzy clustering within the FACE approach. This advancement mitigates the shortcomings of conventional contrast enhancement techniques, minimizing artifacts and suboptimal outcomes. Further enhancing contrast, a universal contrast enhancement variable is ingeniously introduced, enriching image clarity with automatic precision. Experimental validation encompasses a diverse range of yeast cell images subjected to rigorous quantitative assessment via Root-Mean-Square Contrast and Root-Mean-Square Deviation (RMSD). Comparative analyses against conventional enhancement methods showcase the superior performance of the FACE-enhanced images. Notably, the integration of the innovative You Only Look Once (YOLOv5) facilitates automatic cell detection within a finely partitioned grid system. This leads to the development of two models-one operating on pristine raw images, the other harnessing the enriched landscape of FACE-enhanced imagery. Strikingly, the FACE enhancement achieves exceptional accuracy in automatic yeast cell detection by YOLOv5 across both raw and enhanced images. Comprehensive performance evaluations encompassing tenfold accuracy assessments and confidence scoring substantiate the robustness of the FACE-YOLO model. Notably, the integration of FACE-enhanced images serves as a catalyst, significantly elevating the performance of YOLOv5 detection. Complementing these efforts, OpenCV lends computational acumen to delineate precise yeast cell contours and coordinates, augmenting the precision of cell detection.

Effectiveness and safety of human umbilical cord-mesenchymal stem cells for treating type 2 diabetes mellitus.

BACKGROUND: Progressive pancreatic ß-cell dysfunction is a fundamental part of the pathology of type 2 diabetes mellitus (T2DM). Cellular therapies offer novel opportunities for the treatment of T2DM to improve the function of islet ß-cells. AIM: To evaluate the effectiveness and safety of human umbilical cord-mesenchymal stem cell (hUC-MSC) infusion in T2DM treatment. METHODS: Sixteen patients were enrolled and received 1 × 106 cells/kg per week for 3 wk as intravenous hUC-MSC infusion. The effectiveness was evaluated by assessing fasting blood glucose, C-peptide, normal glycosylated hemoglobin A1c (HbA1c), insulin resistance index (homeostatic model assessment for insulin resistance), and islet ß-cell function (homeostasis model assessment of ß-cell function). The dosage of hypoglycemic agents and safety were evaluated by monitoring the occurrence of any adverse events (AEs). RESULTS: During the entire intervention period, the fasting plasma glucose level was significantly reduced [baseline: 9.3400 (8.3575, 11.7725), day 14 ± 3: 6.5200 (5.2200, 8.6900); P < 0.01]. The HbA1c level was significantly reduced on day 84 ± 3 [baseline: 7.8000 (7.5250, 8.6750), day 84 ± 3: 7.150 (6.600, 7.925); P < 0.01]. The patients' islet ß-cell function was significantly improved on day 28 ± 3 of intervention [baseline: 29.90 (16.43, 37.40), day 28 ± 3: 40.97 (19.27, 56.36); P < 0.01]. The dosage of hypoglycemic agents was reduced in all patients, of whom 6 (50%) had a decrement of more than 50% and 1 (6.25%) discontinued the hypoglycemic agents. Four patients had transient fever, which occurred within 24 h after the second or third infusion. One patient (2.08%) had asymptomatic nocturnal hypoglycemia after infusion on day 28 ± 3. No liver damage or other side effects were reported. CONCLUSION: The results of this study suggest that hUC-MSC infusion can improve glycemia, restore islet ß-cell function, and reduce the dosage of hypoglycemic agents without serious AEs. Thus, hUC-MSC infusion may be a novel option for the treatment of T2DM.

How to identify the posterior boundary of No. 8a and the right side group of No. 9 lymph nodes?

As the development of laparoscopic gastrectomy for gastric cancer in recent years, laparoscopic lymphadenectomy becomes more and more feasible and widely accepted. But there're still some issues confuse gastric surgeons, such as the precise extent of the lymph nodes (LN), especially the posterior boundary of No. 8a and the right side group of No. 9 LNs. Here we introduce a reasonable and feasible method to identify the posterior boundary of No. 8a and the right side group of No. 9 LNs and demonstrate the detail procedure.

Reduced tumorigenicity and drug resistance through the downregulation of octamer-binding protein 4 and Nanog transcriptional factor expression in human breast stem cells.

Breast cancer is the most common type of malignancy among females. Previous studies examining breast cancer tissue have demonstrated the presence of stem cells, and have detected octamer­binding protein 4 (Oct4) and Nanog transcription factor expression. In the present study, breast cancer stem cells (CSCs) were isolated and enriched from MDA­MB­231 breast cancer cell lines, and were defined as MDA­MB­231 stem cells using flow cytometry. The expression of Oct4 and Nanog in breast CSCs were detected by quantitative polymerase chain reaction and western blotting. RNA interference (RNAi) was used in order to downregulate the expression of Oct4 and Nanog. Drug resistance and tumor­initiating capability following in vivo injection of MDA­MB­231 stem cells trans-duced with negative RNAi, Oct4 RNAi and Nanog RNAi were compared with that of MDA­MB­231 stem cells without siRNA transfection as a control group. In addition the capability of MDA­MB­231 breast cancer cells to initiate tumor formation in mice was compared with that of MDA­MB­231 stem cells. A paclitaxel inhibition test was also conducted in order to detect resistance of MDA­MB­231 breast cancer stem cells to this treatment. The MDA­MB­231 stem cells were revealed to exhibit elevated percentages of the cluster of differentiation (CD)44+CD24­/low subset, high tumorigenicity and resistance to chemotherapy, all of which are characteristic stem cell properties. In addition, the MDA­MB­231 stem cells were more tumorigenic in vivo. Furthermore, the breast CSCs also expressed high levels of the Oct4 and Nanog transcription factors. Therefore, downregulation of Oct4 or Nanog expression may reduce chemotherapeutic drug resistance and tumorigenicity in breast CSCs. In conclusion, Oct4 and Nanog expression may be a key factor in the development of resistance to chemotherapy and tumor growth of breast CSCs. This finding indicates that Oct4 or Nanog­targeted therapy may be a promising means of overcoming resistance to chemotherapy and inhibiting tumor growth in breast cancer treatment.

[The clinical significance of preoperative enteral immune nutrition in patients with malignant gastrointestinal tumors].

OBJECTIVE: To study the impact of preoperative enteral immune nutrition on patients with malignant gastrointestinal tumors. METHODS: 82 patients with malignant gastrointestinal tumors were divided equally into 2 groups:enteral nutrition group (EN) and normal diet group (Control). Enteral Nutritional Emulsion (TPF-T) served as nasogastically-fed liquid diet for the patients in EN group over a period of 7 days prior to surgery. Normal diet was given to the patients in control group under the same condition as those in EN group in terms of calories and nitrogen contents. Enzyme linked immunosorbent assay (ELISA) was performed to determine the quantity of serum albumin (ALB), transferrin protein (TRF), pre-albumin (PA) and retinol binding protein (RBP). Flow cytometry (FCM) was performed to determine T cell subsets. Postoperative complications, resumption of peristalsis, length of hospital stay, and nutritional costs were also recorded. RESULTS: TRF, PA and RBP increased significantly in the patients in EN group compared with those in control group (P < 0.05). The patients in EN group had significantly higher proportions of CD3+, CD4+/CD8+ higher than those of control (P < 0.05). No serious complications (eg. death or gastrointestinal fistula) were found in the patients. The total nutritional cost for the patients in EN group was similar to that of the controls (P > 0.05). The patients in EN group had less postoperative complications, quicker resumption of peristalsis, shorter hospital stay and lower level of postoperative nutrition cost compared with those of controls (P < 0.05). CONCLUSION: Enteral nutrition support can improve the nutritional status and immunity of patients with malignant gastrointestinal tumors, which has both pre-operative and post-operative benefits for the patients.

[Cytosine deaminase and thymidine kinase double suicide gene system driven by carcinoembryonic antigen promoter for the treatment of colorectal carcinoma xenograft in nude mice].

OBJECTIVE: To explore the therapeutic efficacy of double suicide gene system driven by carcinoembryonic antigen (CEA) promoter (Cp-CDglyTK) on colorectal carcinoma xenograft in nude mice. METHODS: The plasmid pcDNA3.1(-)Cp-CDglyTK was transfected into the CEA-positive SW480 and CEA-negative HeLa cells respectively. The expression of suicide gene was detected by RT-PCR. And the transfected cells were treated with 5-fluorocytosine (5-FC) and ganciclovir (GCV) at different concentrations and the cell-killing and bystander effects assayed by methyl thiazolyl tetrazolium (MTT). By a transplantation of cultivated cells, SW480 or HeLa cell lines were injected subcutaneously into right axillary of nude mice to establish 96 SW480 and 72 HeLa tumor animal models. Nude mice were completely randomized with statistical software according to tumor volume. For prodrug therapy, 48 SW480-bearing mice were divided equally into 4 groups of I-IV. At the same time, 48 HeLa-bearing mice were divided equally into 4 groups of V-VIII. Groups I & V received an intratumoral injection of PBS, groups II & VIGCV and 5-FC intratumorally, groups III & VII PBS intraperitoneally and groups IV & VIII GCV and 5-FC intraperitoneally. Forty-eight SW480-bearing mice were divided equally into 4 groups of IX∼XII and 24 Hela-bearing ones into groups of & in therapy experiment by suicide gene plus prodrug. Six groups received an intratumoral injection of liposome Lipofectamine and plasmid CP-CDglyTK and then an intraperitoneal injection of drug. The groups of IX and received an injection of PBS, group X GCV, group XI 5-FC and groups XII & GCV and 5-FC. The observation parameters included tumor bulk, tumor weight, survival time and treatment effect in each group. RESULTS: SW480 cells transfected by plasmid pcDNA3.1(-)Cp- CDglyTK expressed CDglyTK gene. The inhibition rates of GCV and 5-FC were significantly higher than those of HeLa cells (59.87% ± 0.21% vs 9.90% ± 0.09%, P < 0.01). And higher inhibition rates and stronger bystander effect existed in double versus single produg (all P < 0.05). Tumor size, final tumor weight and survival time of nude mice in groups ofII, IV, VI & VIII had no significant difference with groups ofI, III, V & VII (all P < 0.05). Final tumor size and weight of group XII was significantly smaller than those of groups of IX, X and XI ((150.0 ± 3.2) vs (522.5 ± 1.9) and (256.8 ± 10.4) and (260.7 ± 2.2) mm(3), (54.1 ± 10.4) vs (682.0 ± 12.0) and (251.8 ± 15.1) and (271.6 ± 17.7) mg, all P < 0.05). Meanwhile, the tumor inhibition rate and survival time of group XII(92.1% and (25.7 ± 0.8)d) were significant higher and longer than group X (63.1% and (21.8 ± 0.5) d) and group XI (60.2% and (18.0 ± 0.9) d) (all P < 0.05). However, no significant difference existed in tumor size, final tumor weight and survival time between groups and (all P > 0.05). The inhibition rate of group was merely 0.9%. CONCLUSION: CDglyTK double suicide gene system driven by CEA promoter may inhibit CEA positive colorectal cancer xenograft in prodrug-treated nude mice.

Targeting the vasculature of colorectal carcinoma with a fused protein of (RGD)3-tTF.

PURPOSE: Truncated tissue factor (tTF) fusion protein targeting tumor vasculature can induce tumor vascular thrombosis and necrosis. Here, we generated (RGD)3-tTF in which three arginine-glycine-aspartic (RGD) targeting integrin α(v)ß3 and tTF induce blood coagulation in tumor vessels. METHODS: The bioactivities of (RGD)3-tTF including coagulation activity, FX activation, and binding with integrin α(v)ß3 were performed. The fluorescent labeled (RGD)3-tTF was intravenously injected into tumor-bearing mice and traced in vivo. The tumor growth, volume, blood vessel thrombosis, tumor necrosis, and survival time of mice treated with (RGD)3-tTF were evaluated. RESULTS: The clotting time and FX activation of (RGD)3-tTF were similar to that of TF (P > 0.05) but different with that of RGD (P < 0.05). (RGD)3-tTF presented a higher binding with α(v)ß3 than that of RGD and TF at the concentration of 0.2 µmol/L (P < 0.05). (RGD)3-tTF could specifically assemble in tumor and be effective in reducing tumor growth by selectively inducing tumor blood vessels thrombosis and tumor necrosis which were absent in mice treated with RGD or TF. The survival time of mice treated with (RGD)3-tTF was higher than that of mice treated with TF or RGD (P < 0.05). CONCLUSION: (RGD)3-tTF may be a promising strategy for the treatment of colorectal cancer.

Radicicol, an Hsp90 inhibitor, inhibits intestinal inflammation and leakage in abdominal sepsis.

BACKGROUND: Intestinal injury is a key feature in sepsis. Inhibitors of heat shock protein 90 (Hsp90) have been shown to exert protective effects in models of inflammation. Herein, we hypothesized that Hsp90 might regulate intestinal inflammation and leakage in abdominal sepsis. MATERIALS AND METHODS: Male C57BL/6 mice were pretreated with radicicol (60 mg/kg), which is a specific inhibitor of Hsp90, prior to cecal ligation and puncture (CLP). Intravital fluorescence microscopy was used to quantify leukocyte-endothelium interactions in the colonic microcirculation 6 h after CLP. Colonic tissue was harvested to determine levels of myeloperoxidase, tumor necrosis factor-α and CXC chemokines. Intestinal injury was examined by histology. Intestinal barrier function was quantified by leakage of fluorescein isothiocyanate-dextran from the vascular system out into the abdominal cavity after intravenous injection. RESULTS: We found that radicicol significantly decreased CLP-induced leukocyte rolling and adhesion in colonic venules. Inhibition of Hsp90 reduced colonic levels of myeloperoxidase by 24% in septic animals. Moreover, radicicol significantly decreased CLP-provoked formation of CXC chemokines but had no significant effect on tumor necrosis factor-α levels in the colon. Notably, Hsp90 inhibition significantly attenuated intestinal tissue injury evoked by CLP. Lastly, it was found that radicicol reduced sepsis-induced intestinal leakage by 43%. CONCLUSION: Our novel findings suggest that targeting Hsp90 protects against intestinal inflammation and leakage and might be a useful strategy to ameliorate intestinal failure in polymicrobial sepsis.

[Humanized monoclonal antibody TNT-3-mediated truncated tissue factor for the treatment of H22 hepatoma-bearing mice].

OBJECTIVE: To investigate the inhibitory effects of humanized monoclonal antibody-3 (huTNT-3) mediated truncated tissue factor (tTF) on the H(22) hepatoma-bearing mice, and to explore its mechanisms. METHODS: The coagulation activity of the huTNT-3/tTF fusion protein was detected by clotting assay and clotting factor X (FX) activation test in vitro. Mouse hepatoma cell line H(22) cells were inoculated subcutaneously into mice to establish the mouse models of hepatoma. The mice were randomly divided into two groups to be injected once with huTNT-3/tTF fusion protein or tTF protein labeled with rhodamine B isothiocyanate (RBITC), respectively. The localization of huTNT-3/tTF fusion protein in the mouse hepatoma tissue was analyzed by confocal laser scanning microscopy 24 hour after the injection. Fifteen mice were randomly divided into three groups to be injected with the huTNT-3/tTF fusion protein, tTF protein or phosphate buffered saline (PBS) once, respectively. The tumor size was measured every two days to calculate the tumor volume. Ten days after the injection the mice were sacrificed. Samples of the tumor, heart, livers, spleen, lung, kidney and brains of the mice were taken for histopathological examination. RESULTS: Both the huTNT-3/tTF fusion protein and tTF protein effectively promoted blood coagulation. Under the conditions of Ca(2+), the coagulation time in the 1.5, 3, 6 µmol/L huTNT-3/tTF groups was (12.90 ± 0.60) min, (10.39 ± 0.40) min and(8.15 ± 0.24) min, respectively, and the coagulation time of the 1.5, 3, 6 µmol/L tTF groups was (14.23 ± 0.46) min, (12.10 ± 0.49) min and (9.83 ± 0.52) min, respectively, the difference between the two groups was not significant (F = 0.145, P = 0.705). The huTNT-3/tTF fusion protein was similar to the tTF protein in the ability of activating FX (t = 0.101, P > 0.05). The confocal laser scanning microscopic analysis showed that RBITC-fluorescence labeled huTNT-3/tTF fusion protein was enriched in the hepatoma tissue. The tumor volume of the huTNT-3/tTF fusion protein group was significantly lower than that of the tTF and PBS groups (both P < 0.001), however, there was not significant difference between the tTF and PBS groups (t = -0.616, P > 0.05). The survival time of the huTNT-3/tTF group was (25.5 ± 2.5) d, significantly longer than that of the PBS group (17.3 ± 1.9) d and the tTF group (18.6 ± 1.9) d, (both P < 0.05). CONCLUSION: The huTNT-3/tTF fusion protein retains the coagulation ability and has the capability of targeting to tumor vasculature, and induces thrombosis in the tumor vessels, thus to suppress the growth of hepatoma in the mice.

[Expression of OCT4 in gastric cancer cell lines and its significance].

OBJECTIVE: To detect the expression of octamer-binding protein-4 (OCT4) in gastric cancer cell lines with different differentiation (MKN-28, SGC-7901, BGC-823) and normal gastric mucosal cells line GES-1, and further assess the relationship between OCT4 expression and the differentiation grade of gastric carcinoma cells. METHODS: Expression level of OCT4 in GES-1, MKN-28, SGC-7901 and BGC-823 was detected by reverse transcription-polymerase chain reaction (RT-PCR), and OCT4 siRNA was employed to interfere OCT4 expression in BGC-823 cell lines. Detect the quantity of OCT4 mRNA and OCT4 protein by fluorescent quantitative PCR and Western blot respectively. In addition, the invasion ability was analyzed via Transwell chamber. RESULTS: The normal gastric mucosal cells did not express OCT4 and there was higher expression of OCT4 in gastric cancer cell lines with poorly differentiation (P<0.05). The expression of OCT4 in BGC-823 cells was the highest. The expression of OCT4 mRNA and OCT4 protein were decreased distinctly in BGC-823 cells after being interfered by OCT4 siRNA (P<0.05). After being interfered by OCT4, BGC-823 cells were less aggressive, and the number of penetrating cells was decreased (P<0.05). CONCLUSION: The OCT4 expression level is associated with gastric cancer differentiation. OCT4 may play an important role in the differentiation and invasion of gastric cancer cell and it may serve as a reference index in predicting the malignant grade of gastric cancer.

[Immune tolerance induced by bone marrow cell transplantation combined with use of cyclophosphamide in diabetic rats with pancreatic transplantation].

OBJECTIVE: To study the immune tolerance induced by bone marrow cell transplantation combined with short-term use of cyclophosphamide after pancreatic transplantation in diabetic rats. METHODS: Type I diabetes mellitus was induced in BN rats with streptozotocin (STZ) intraperitoneal injection at a single dose of 45 mg/kg. Pancreatic transplantations were performed with the SD rats as donors and the diabetic BN rats as recipients. Twenty BN rats with type I diabetes mellitus were randomly divided into four groups. The BN rats in Group I received pancreas transplantations only. The BN rats in Group II received intraperitoneal injection of 150 mg/kg cyclophosphamide on the first day after pancreas transplantations. The BN rats in Group 11 received injection of 2.0 x 10(8) donors' bone marrow cells via the portal vein during the pancreas transplantations. The BN rats in Group IV received injection 2.0 x 10(8) donors' bone marrow cells via the portal vein during the pancreas transplantations and an intraperitoneal injection of 150 mg/kg cyclophosphamide on the first day after pancreas transplantations. The blood glucose of the rats was measured after transplantations. The graft functional survival time (GFST) was recorded. Peripheral blood was obtained two weeks after the transplantations to prepare single cell suspension for detecting chimera formation rate and the level of Vbeta11+ T cell by flow cytometry. RESULTS: The average GFST of group IV was (18 +/- 2.2) d, significantly longer than those of group I (7.8 +/- 1.2) d, group II (8.2 +/- 1.6) d, and group III (8.8 +/- l.4) d (P < 0.05). The rats in group IV had significant lower level of Vbeta11+ T cells (2.5 +/- 0.3)% than those in the other groups (P < 0.05). Donors' bone marrow-derived cells could be detected in the peripheral blood of diabetic rats in group IV, with a chimeric rate of (10.0 +/- 2.3)%. No donors' bone marrow-derived cells were detected in the rats in other groups. CONCLUSION: Bone marrow cell transplantation combined with short-term use of cyclophosphamide promote chimerism formation and induce immune tolerance in rats with pancreatic transplantations, which prolongs pancreatic graft functional survival time.

[Arginine-glycine-aspartic polypeptide mediated truncated tissue factor therapy for colorectal carcinoma].

OBJECTIVE: To explore the therapy effects of (arginine-glycine-aspartic, RGD)(3)-truncated tissue factor (tTF) fusion protein on colorectal carcinoma in mice. METHODS: The (RGD)(3)-tTF fusion gene, constructed with tTF and three series-wound peptides RGD, was expressed in Escherichia coli BL21 (DE(3)). The fusion protein was purified through Nickel affinity chromatography column. The coagulation activity of the (RGD)(3)-tTF fusion protein was detected by clotting assay in vitro. Mice colorectal cancer cells line CT26 were inoculated subcutaneously into mice to establish colorectal cancer model. Four mice were randomly divided into two groups to be injected with the (RGD)(3)-tTF or tTF fusion protein labeled with rhodamine B isothiocyanate (RBITC) at a single dose of 50 microg respectively. The location of the (RGD)(3)-tTF fusion protein in the colorectal carcinoma bearing mice tissue was analyzed by using in vivo optical imaging one hour after the injection and confocal microscopy twenty-four hours after the injection. Fifteen mice bearing colorectal carcinoma were randomly divided into three groups for injection with the (RGD)(3)-tTF, tTF fusion protein or phosphate buffered saline (PBS) at a single dose of 50 microg respectively. The tumor size was measured daily to calculate the tumor volume. Five days after the injection, the mice were killed to harvest tumor tissues, hearts, livers, spleens, lung, kidneys and brains to observe valid thrombogenesis and tumor necrosis. RESULTS: With the concentration of the (RGD)(3)-tTF fusion protein increased, the clotting time was shorten correspondingly under the conditions of Ca(2+), and the clotting time was (8.6 +/- 0.2) min when the concentration was 6 micromol/L, and it was >30 min in the group of 0 micromol/L (P < 0.05). The coagulation activity of (RGD)(3)-tTF and tTF fusion protein was alike (F = 0.09, P > 0.05). The in vivo optical imaging and confocal microscopy analyses showed that RBITC fluorescence labeling (RGD)(3)-tTF fusion protein was assembled in the tumor vasculature. On the first, third, fifth day after injection, the tumor volume of (RGD)(3)-tTF fusion protein group was (120.8 +/- 4.8) mm(3), (93.8 +/- 3.4) mm(3), (132.2 +/- 7.7) mm(3) respectively, which was significantly smaller than that of the tTF group [(181.4 +/- 13.8) mm(3), (333.0 +/- 32.0) mm(3), (514.0 +/- 11.5) mm(3)] and PBS group [(182.6 +/- 11.5) mm(3), (332.8 +/- 21.0) mm(3), (524.2 +/- 16.7) mm(3)] (both P < 0.05). However, there was no significant difference in the tumor volume between the latter two groups (P > 0.05). CONCLUSION: The (RGD)(3)-tTF fusion protein is capable of targeting to tumor vasculature and inducing thrombogenesis for suppressing the tumor growth in the colorectal carcinoma mice model, and it's expected to be a new therapy for colorectal cancer.