[Effect of SIRT7 on inhibiting the epithelial-mesenchymal transformation in pancreatic cancer cells and related mechanism].

Objective: To investigate the effect and mechanism of SIRT7 in epithelial mesenchymal transformation (EMT) of pancreatic cancer cells. Methods: The pancreatic cancer cells were divided into siControl, siSIRT7, over-expression SIRT7, siSIRT7+siCOL4A1, and siSIRT7+siSLUG groups using siRNA or plasmid transfection. The proliferation, migration and invasion of pancreatic cancer cells were detected by EdU, wound healing assay and Transwell experiments, respectively. The expression of EMT and cancer stem cell (CSC) markers were detected by quantitative real-time reverse transcription polymerase chain reaction assay (qRT-PCR) and western blot. RNA sequencing (RNA-seq) in SIRT7 knockdown PANC-1 cells was performed to explore the signaling pathways and target genes regulated by SIRT7. Then the target genes directly regulated by SIRT7 were identified with quantitative chromatin immunoprecipitation experiment (q-ChIP) and chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR). The expressions of SIRT7 and target genes were detected by immunohistochemical (IHC) in pancreatic cancer tissues, and the correlation between SIRT7 and target gene expression was analyzed using TCGA dataset. The correlation between expression of SIRT7 or target genes and survival was analyzed on KM-plotter website. Finally, GeneMANIA, STRING and ENCORI were used to predict SIRT7-related proteins and miRNAs. Results: EdU assay showed that the cell proliferation rates in SIRT7-overexpressed PANC-1 [(19.33±0.35)%] and BxPC-3 cells [(17.00±1.89)%] were lower than those in the control group [(31.60±1.37)% and (24.33±0.78)%, respectively, P＜0.05]. The proliferation rates of SIRT7-knockdown PANC-1 [(23.94±1.00)% and (27.08±0.97)%] and BxPC-3 cells [(22.00±1.86)% and (25.96±1.61)%] were higher than those of the siControl group [(11.80±1.86)% and (13.42±1.39)%, respectively, P＜0.05]. In PANC-1 cells, the wound healing assay showed that the relative migration rate of SIRT7-overexpression cells [(76.67±2.74)%] was lower than that of control cells [(100.00±2.13)%, P＜0.05]; the relative migration rate of cells with SIRT7 knockdown [(134.22±4.08)% and (199.82±9.20)%, respectively] was higher than that of siControl group [(102.24±3.13)%, P＜0.05]. Compared with the control group, SIRT7 overexpression decreased the number of migrated BxPC-3 cells (45.66±1.69 vs 28.33±2.62, P＜0.05); while SIRT7 knockdown increased these numbers (65.66±2.86 and 82.00±2.94 versus 33.00±0.81, P＜0.01). Transwell experiment revealed that the number of invaded cells in SIRT7 overexpression groups (16.33±2.05 and 34.66±1.69) was lower than that control groups (54.33±4.64 and 58.66±5.90, P＜0.05); with SIRT7 knockdown, the numbers of invaded PANC-1 (63.66±2.49 and 69.33±3.29) and BxPC-3 cells (134.33±3.09 and 181.66±4.02) were higher than those in control groups (35.33±2.49 and 42.00±0.81, P˂0.05). Also, SIRT7 knockdown decreased the expressions of epithelial markers and increased the expressions of mesenchymal and CSC markers. RNA-seq analysis showed that SIRT7 was involved in regulating a variety of cancer-related signaling pathways, including the pancreatic cancer pathway and the EMT pathway. Furthermore, SIRT7 could directly bind to the promoter regions of target genes, such as COL4A1 and SLUG. SIRT7 was negatively correlated with the expression and function of COL4A1 and SLUG in pancreatic cancer cells. The expressions of SIRT7, COL4A1, SLUG and SOX2 were verified in pancreatic cancer tissues by IHC. Finally, SIRT7 was predicted to be associated with many proteins and miRNAs based on GeneMANIA, STRING, and ENCORI online tools. Conclusions: SIRT7 can inhibit the EMT of pancreatic cancer cells through transcriptionally inhibiting the expression of target genes, such as COL4A1 and SLUG. Thus, SIRT7 may serve as a potential tumor suppressor gene in pancreatic cancer.

[Precise minimally invasive treatment of mandibular fracture in children assisted by digital surgery technology].

Objective: To explore the role of digital three-dimensional printing technology and multifunctional board in the treatment of mandibular fracture in children. Methods: From January 2006 to January 2022, 42 children with mandibular fracture were treated by Department of Stomatology, in The First Affiliated Hospital of Bengbu Medical College, including 25 males and 17 females. The patients, aged from 4 to 12 years, with the median age was 10 years old, were divided into observation group (22 cases) and routine group (20 cases) according to the treatment methods. In the observation group, the multifunctional board was made before operation, and the CT data of the children were imported into Mimics software in".dicom"format, and the displaced mandible was virtually reset. The jaw reduction model was made by three-dimensional printing, and the surgical operation was simulated on the reduction model to determine the model and position of the internal fixation device and shape it. During the operation, the fracture was reduced and fixed according to the preoperative design; The conventional group was treated with open reduction and internal fixation of mandibular fracture by traditional methods, and the clinical application value was compared and analyzed through the intraoperative situation, occlusal relationship, and follow-up of the two groups. Results: The total intraoperative bleeding volume [(30.25±4.02) ml] and surgical time [(64.3±9.2) min] in the observation group were significantly lower than those in the conventional group [(35.13±5.69) ml and (84.6±13.9) min, respectively] (F=6.18, P=0.003; F=1.32, P=0.001). The excellent and good rate of occlusal relationship in the observation group [96% (21/22)] was significantly higher than that in the conventional group [85% (17/20)] (F=4.27, P=0.039). The incidence of complications, the observation group, 1 case of poor occlusion, 1 case of postoperative infection; In the routine group, there were 3 cases with poor occlusion, 1 case with nerve injury, 1 case with root injury and 1 case with tooth germ injury. Conclusions: The application of digitization three-dimensional printing technology combined with multifunctional occlusal plate in children's mandibular fracture is minimally invasive, safe, efficient and accurate, and the clinical effect is good.

Effects of the circadian rhythm on milk composition in dairy cows: Does day milk differ from night milk?

Metabolism in most organisms can show variations between the day and night. These variations may also affect the composition of products derived from livestock. The aim of the present study was to investigate the difference in composition between the day milk and night milk of dairy cows. Ten multiparous Holstein cows (milk yield = 25.2 ± 5.00 kg/d) were randomly selected during mid lactation. Milk samples were collected at 0500 h ("night milk") and 1500 h ("day milk") and analyzed to determine their composition. Mid-infrared spectroscopy was used to analyze macronutrient content of milk. Metabolomics and lipidomics were used to detect and analyze small molecules and fatty acids, respectively. An automatic biochemical analyzer and ELISA kits were used to determine biochemical indicators, as well as antioxidant and immune parameters in the milk. Though milk fat, protein, lactose, and total milk solids were not different between day milk and night milk, small molecules, metabolites and lipids, and hormones and cytokines differed between day milk and night milk. Regarding biochemical and immune-related indicators, the concentrations of malondialdehyde, HSP70, and HSP90 in night milk were lower than that in day milk. However, interferon-γ levels were higher in night milk. Additionally, night milk was naturally rich in melatonin. Lipidomics analyses showed that the levels of some lipids in night milk were higher than those in day milk. Metabolomics analyses identified 36 different metabolites between day milk and night milk. Higher concentrations of N-acetyl-d-glucosamine, cis-aconitate, and d-sorbitol were observed in day milk. However, the other 33 metabolites analyzed, including carbohydrates, lipids, AA, and aromatic compounds, showed lower concentrations in day milk than in night milk. The present findings show that the composition of night milk differs considerably from that of day milk. Notable changes in the circadian rhythm also altered milk composition. These results provide evidence to support the strategic use and classification of day milk and night milk.

[Application of superficial inferior epigastric artery flaps to repair oral and maxillofacial defects with the aid of digital three-dimensional reconstruction technique].

Objective: To explore the application value of digital three-dimensional(3D) reconstruction technology in the repair of oral and maxillofacial defects with superficial inferior epigastric artery (SIEA) flap. Methods: Twelve cases of oral cancer patients, including 8 males and 4 females; aged (57.4±12.6) years, were selected from the Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Bengbu Medical College from January 2018 to October 2019 and were proposed to repair with SIEA flap. There were 10 cases of squamous cell carcinoma, one case of adenoid cystic carcinoma and 1 case of mucinous epidermal carcinoma. The data were imported into AW4.7 software for post-processing. The left or right dominant donor area was selected to clarify the origin, diameter, alignment, and location of penetration point of the flap blood supply, and digital 3D reconstruction technology was used to guide the flap preoperative design. Results: Eleven cases were repaired by SIEA flap in 12 patients, one case was repaired by superficial iliac artery flap because the source artery was undiscovered, one case had venous vascular crisis after surgery, and the rest of the flap survived. In 11 patients repaired with SIEA flap, there was no significant difference between the preoperative SIEA diameter measured by CTA [(1.0±0.3) mm] and the actual measured value [(1.1±0.3) mm] (P>0.05). The follow-up was 6 to 12 months, with an average of 10 months, and the donor-receiver areas were all healed in phase Ⅰ. No obvious complications occurred, and the abdominal scar was hidden. Conclusions: In the SIEA flap repair oral and maxillofacial defect reconstruction surgery, the use of digital 3D reconstruction technology can objectively reflect the diameter and the location of the superficial artery of the abdominal wall before surgery, effectively reduce the difficulty and risk of flap surgery.

Overexpression of SREBF chaperone (SCAP) enhances nuclear SREBP1 translocation to upregulate fatty acid synthase (FASN) gene expression in bovine mammary epithelial cells.

Fatty acid synthase is a key enzyme for the synthesis of milk fat in the ruminant mammary gland. In nonruminants, sterol regulatory element binding protein 1 (SREBP1) is a regulator of FASN gene expression, and SREBF chaperone (SCAP) is essential for SREBP1 maturation and activity. However, the role of SCAP on the regulation of FASN gene expression in ruminants is unknown. The objective of this study was to investigate the transcriptional regulation of FASN by overexpressing SCAP in bovine mammary epithelial cells. A bovine SCAP expression vector, SREBP1 expression vector, and the promoter of FASN were cloned. The transcription factor binding sites of FASN promoter were predicted using bioinformatics analysis. After transfection with FASN promoter vectors in the immortalized bovine mammary epithelial cell line MAC-T, we co-overexpressed the SCAP + SREBP1 expression vector with pcDNA3.1 vector as control. The effect of SCAP + SREBP1 overexpression on the regulation of FASN was investigated using luciferase assay, immunofluorescence, Western blot, real-time PCR, and lipid droplet staining. We observed that co-overexpression of SCAP + SREBP1 significantly increased activity of the FASN promoter containing a sterol response element binding site. The FASN mRNA abundance and lipid droplet formation increased due to co-overexpression of SCAP + SREBP1. Compared with overexpression of SREBP1 alone, co-overexpression of SCAP + SREBP1 enhanced the nuclear translocation and nuclear SREBP1 protein abundance. Overall, as in nonruminants cells, results indicate that SCAP is essential for promoting nuclear translocation of SREBP1 and activation of FASN gene transcription, leading to lipid droplet formation in bovine mammary epithelial cells.

Two mutations in the 5'-flanking region of the KITLG gene are associated with litter size of dairy goats.

In this study, Xinong Saanen (SN) and Guanzhong (GZ) dairy goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the 5'-flanking region of the KITLG gene by DNA sequencing and primer-introduced restriction analysis-polymerase chain reaction. Two novel SNPs (g.13090G>T and g.13664C>A) were identified (GenBank Accession no. KM658964). Furthermore, g.13090G>T and g.13664C>A loci were closely linked in SN and GZ breeds (r(2) > 0.33). Association analysis results showed that g.13090G>T and g.13664C>A SNPs significantly affected litter size (P < 0.05). The litter size of individuals with the combined genotype GG/CC from both dairy goat breeds was greater than that of individuals with TT/AA in average parity (P < 0.05). Known biochemical and physiological functions, along with our results, indicated that GG/CC could be used in marker-assisted selection to choose individuals with greater litter size from both breeds. These results extend the spectrum of genetic variation in the caprine KITLG gene and may contribute to genetic resources and breeding of goats.

Polymorphism and DNA methylation in the promoter modulate KISS1 gene expression and are associated with litter size in goats.

Polymorphisms in the promoter region are likely to impact KISS1 gene transcription and reproductive traits. In this study, Guanzhong (GZ, n=350) and Boer (BE, n=196) goats were used to detect polymorphism in the promoter of the goat KISS1 gene by DNA sequencing. In the GZ goats, the g.1384G>A mutation was identified in the promoter of the goat KISS1 gene. Guanzhong goats were in Hardy-Weinberg disequilibrium at g.1384G>A locus (P<0.05). The 1384A allele was predicted to eliminate methylation, AHR-arnt heterodimers and AHR-related factors (AHRR) and myoblast determining factors (MYOD) transcription factor-binding sites. Statistical results indicated that the g.1384G>A SNP was associated with litter size in the GZ goats (P<0.05). Luciferase assay analysis suggested that the 1384A allele increased luciferase activity when compared to the 1384G allele. The RT-qPCR assay also demonstrated that the 1384A allele had greater amounts of KISS1 mRNA than the 1384G allele in homozygous individuals. Functional analysis suggested that this g.1384G>A SNP may be an important genetic regulator of KISS1 gene expression with effects on downstream processes that are modulated by KISS1 gene because of the changes of methylation and transcription factor-binding sites. Therefore, the current study provides evidence in goats for genetic markers that might be used in breeding programs.

Association analysis between variants in KITLG gene and litter size in goats.

Xinong Saanen (SN) and Guanzhong (GZ) goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the coding regions with their intron-exon boundaries and the proximal flanking regions of KITLG gene by DNA sequencing and genotyped by PCR-restriction fragment (PCR-RFLP). Four novel SNPs (g.12654G>A, g.12772G>A, g.12829T>C and g.23683C>T) were identified (GenBank accession No. KM609289). It was shown that Xinong Saanen and Guanzhong goat breeds were in Hardy-Weinberg disequilibrium at g.12654G>A, g.12772G>A and g.12829T>C loci (P<0.05). The g.12654G>A, g.12772G>A and g.12829T>C loci were closely linked in both goat breeds (r(2)>0.33). Results of an association analysis indicated that SNPs g.12654G>A, g.12772G>A and g.12829T>C had significant effects on litter size (P<0.05). The combined genotypes of four SNP loci also affected litter size with the C7(GG/GG/CC/CC) genotype in the SN goat breed and C1(AA/GG/CC/CC) and C7(GG/GG/CC/CC) genotypes in the GZ goat breed having the highest litter size. The biochemical and physiological functions, together with the results obtained in our investigation, suggest that C7(GG/GG/CC/CC) could be used in marker-assisted selection to select the individuals with higher litter size in both goat breeds. The results extend the spectrum of genetic variation of the caprine KITLG gene, which might contribute to goat genetic resources and breeding.

Polymorphisms of the ATP1A1 gene associated with mastitis in dairy cattle.

Mastitis affects the concentrations of potassium and sodium in milk. Since sodium-potassium adenosine triphosphatase (Na(+), K(+)-ATPase) is critical for maintaining the homeostasis of these two ions, and is involved in cell apoptosis and pathogenesis, we presumed that polymorphism of the ATP1A1 gene, which encodes the bovine Na(+), K(+)-ATPase α1 subunit could be associated with mastitis. The ATP1A1 gene was analyzed in 320 Holstein cows using PCR low ionic strength single-strand conformation polymorphism (PCR-LIS-SSCP) and DNA sequencing methods. A C/A SNP was identified at nucleotide position -15,739 in exon 17 of the ATP1A1 gene, but it did not induce any change in amino acids. We examined a possible association of polymorphism of the ATP1A1 gene with somatic cell score and 305-day milk yields. Individuals with genotype CC in ATP1A1 had significantly lower somatic cell scores and 305-day milk yields than those with genotype CA. We also examined changes in Na(+), K(+)-ATPase activity of red cell membranes. The Na(+), K(+)-ATPase activity was significantly higher in dairy cows with genotype CC compared to the other two genotypes, and the Na(+), K(+)-ATPase activity of the resistant group was significantly higher than that of the susceptible group in dairy cows. We conclude that this polymorphism has potential as a marker for mastitis resistance in dairy cattle.

Discoloration of methylene blue and wastewater from a plant by a Fe/Cu bimetallic system.

Using a Fe/Cu bimetallic system (Fe/Cu system), the discoloration of both methylene blue in aqueous solution and the colored wastewater from a plant was investigated under the anaerobic condition in batch or continuous reactors. Results show that the Fe/Cu system effectively removed the color with over 88% of color removal efficiency for both methylene blue solution and the wastewater from the plant in batch test. Color removal efficiencies increased rapidly with Fe/Cu dosage and reaction time, respectively, at initial time and slowly to stable values. Optimum pH was neutral range. In addition, in continuous test it also removed the color of the wastewater from the plant with 63% of discoloring efficiency under the condition of 2 h of hydraulic retention time and neutral range of pH (7.0-8.3). High discoloring efficiencies with low chemical oxygen demand removal efficiencies were found in all experiments. The reduction of chromophores in pollutants was the main mechanism of the discoloration in the Fe/Cu system.

Simultaneous nitrogen and phosphorus removal under low dissolved oxygen conditions.

A full-scale test was operated by using low dissolved oxygen activated sludge process to enhance biological nitrogen and phosphorus removal. When the influent concentrations of CODcr, TN and TP varied in a range of 352.9 mg/L-1338.2 mg/L, 34.4 mg/L-96.3 mg/L, and 2.21 mg/L-24.0 mg/L, the average removal efficiencies were 94.9%, 86.7% and 93.0%, respectively. During the test period of two months, effluent means of CODcr, BOD5, NH3-N, TN and TP were below 50 mg/L, 25 mg/L, 10 mg/L and 1.0 mg/L, respectively. The low dissolved oxygen activated sludge process has a simple flow sheet, fewer facilities and high N and P removal efficiency. It is very convenient to retrofit the conventional activated sludge process with the above process.

Membrane bioreactor process of organic wastewater from brassylic acid manufacturing plant.

The wastewater treatment from brassylic acid manufacturing plant using membrane bioreactor (MBR) was studied. The membrane bioreactor consisted of batch-operation biological aeration tank and ultrafiltration evaluation tank. The content of test included the affection of variation operation conditions on ultrafiltration separation, the general characteristics of MBR process, and the difference comparing with the conventional biological treatment. The results are as follows: (1) among the test membrane material, polyether sulphone (PES) membrane is more suitable for the wastewater treatment; (2) when the cutoff molecular weight is among 10,000-50,000, the higher the cutoff molecular weight, the bigger the water flux is in the test; (3) under the operation pressure, water flux increases accompanying with the increasing of operation pressure; (4) the paper filtered COD concentration has more affection on the water flux than the suspended solid concentration; (5) as the volume loading of MBR increases, the accumulation of high molecule organic substance and colloid increases, the membrane permeate COD concentration and paper filtered COD concentration increase too, meanwhile the water flux reduces; (6) when the sludge retention time of activated sludge of MBR increases, the accumulation of high molecule organic substance and colloid reduces, the membrane permeate COD concentration and paper filtered COD concentration reduce too, and the water flux increases; (7) comparing with the conventional biological process, the microbial activity is higher, but the microbial species is less.