Mechanical Behavior of 3D-Printed Thickness Gradient Honeycomb Structures.

In order to obtain a lightweight, high-strength, and customizable cellular structure to meet the needs of modern production and life, the mechanical properties of four thickness gradient honeycomb structures were studied. In this paper, four types of honeycomb structure specimens with the same porosity and different Poisson's ratios were designed and manufactured by using SLA 3D-printing technology, including the honeycomb, square honeycomb, quasi-square honeycomb, and re-entrant honeycomb structures. Based on the plane compression mechanical properties and failure mode analysis of these specimens, the thickness gradient is applied to the honeycomb structure, and four structural forms of the thickness gradient honeycomb structure are formed. The experimental results show that the thickness gradient honeycomb structure exhibits better mechanical properties than the honeycomb structure with a uniform cellular wall thickness. In the studied thickness gradient honeycomb structure, the mechanical properties of the whole structure can be significantly improved by increasing the thickness of cell walls at the upper and lower ends of the structure. The wall thickness, arrangement order, shape, and Poisson's ratio of the cell all have a significant impact on the mechanical properties of the specimens. These results provide an effective basis for the design and application of cellular structures in the future.

Association of miR-1247-5p expression with clinicopathological parameters and prognosis in breast cancer.

Our study aimed to clarify the correlation between miR-1247-5p expression and clinicopathological parameters and survival of patients with breast cancer (BC). We evaluated the expression level of miR-1247-5p in 224 formalin-fixed, paraffin-embedded specimens (112 BC and matched cancer free tissues) by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). miR-1247-5p expression in BC tissues was found to be decreased compared with matched normal tissues (P < 0.01). Additionally, low miR-1247-5p expression in BC tissues was significantly associated with the advanced TNM stage (P = 0.007), lymph node metastasis (P = 0.015), poorer pathological differentiation (P = 0.005) and molecular subtype (P = 0.027). The patients in the low miR-1247-5p group had a shorter disease-free survival and overall survival than those in the high miR-1247-5p group (P < 0.01). Furthermore, the univariate and the multivariate analyses showed that miR-1247-5p expression was an independent predictor of overall survival (P < 0.01). Our study showed that miR-1247-5p was related to the biological behaviour of breast tumour and prognosis of patients with BC. miR-1247-5p could be a novel tumour suppressor and act as a potential biomarker and therapeutic agent for breast carcinoma.

Depression of biofilm formation and antibiotic resistance by sarA disruption in Staphylococcus epidermidis.

AIM: To study the effects of disruption of sarA gene on biofilm formation and antibiotic resistance of Staphylococcus epidermidis (S. epidermidis). METHODS: In order to disrupt sarA gene, the double-crossover homologous recombination was applied in S. epidermidis RP62A, and tetracycline resistance gene (tet) was used as the selective marker which was amplified by PCR from the pBR322 and inserted into the locus between sarA upstream and downstream, resulting in pBT2delta sarA. By electroporation, the plasmid pBT2delta sarA was transformed into S. epidermidis. Gene transcription was detected by real-time reverse transcription-PCR (RT-PCR). Determination of biofilm was performed in 96-well flat-bottomed culture plates, and antibiotic resistance was analyzed with test tube culture by spectrophotometry at 570 nm respectively. RESULTS: A sarA disrupted strain named S. epidermidis RP62Adelta sarA was constructed, which was completely defective in biofilm formation, while the sarA complement strain RP62Adelta sarA (pHPS9sarA) restored the biofilm formation phenotype. Additionally, the knockout of sarA resulted in decreased erythromycin and kanamycin resistance of S. epidermidis RP62A. Compared to the original strain, S. epidermidis RP62Adelta sarA had an increase of the sensitivity to erythromycin at 200-400 microg/mL and kanamycin at 200-800 microg/mL respectively. CONCLUSION: The knockout of sarA can result in the defect in biofilm formation and the decreased erythromycin and kanamycin resistance in S. epidermidis RP62A.

Integration of E. coli aroG-pheA tandem genes into Corynebacterium glutamicum tyrA locus and its effect on L-phenylalanine biosynthesis.

AIM: To study the effect of integration of tandem aroG-pheA genes into the tyrA locus of Corynebacterium glutamicum (C. glutamicum) on the production of L-phenylalanine. METHODS: By nitrosoguanidine mutagenesis, five p-fluorophenylalanine (FP)-resistant mutants of C.glutamicum FP were selected. The tyrA gene encoding prephenate dehydrogenase (PDH) of C.glutamicum was amplified by polymerase chain reaction (PCR) and cloned on the plasmid pPR. Kanamycin resistance gene (Km) and the P(BF) -aroG-pheA-T (GA) fragment of pGA were inserted into tyrA gene to form targeting vectors pTK and pTGAK, respectively. Then, they were transformed into C.glutamicum FP respectively by electroporation. Cultures were screened by a medium containing kanamycin and detected by PCR and phenotype analysis. The transformed strains were used for L-phenylalanine fermentation and enzyme assays. RESULTS: Engineering strains of C.glutamicum (Tyr(-)) were obtained. Compared with the original strain, the transformed strain C. glutamicum GAK was observed to have the highest elevation of L-phenylalanine production by a 1.71-fold, and 2.9-, 3.36-, and 3.0-fold in enzyme activities of chorismate mutase, prephenate dehydratase and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, respectively. CONCLUSION: Integration of tandem aroG-pheA genes into tyrA locus of C. glutamicum chromosome can disrupt tyrA gene and increase the yield of L-phenylalanine production.

Regulation of aroP expression by tyrR gene in Escherichia coli.

tyrR gene encodes a global regulatory protein (TyrR), which plays an important role in the transcriptional regulation of eight transcription units (including tyrR gene itself) whose protein products catalyze key steps in aromatic amino acid biosynthesis and/or transport. The aroP gene encodes an integral membrane protein (AroP) that transports aromatic amino acids through the cell membrane. The transcription of aroP was reported to be repressed by TyrR. In this work, aroP(p) (aroP gene carrying its own promoter), aroP (aroP gene without promoter) and tyrR genes were amplified by PCR from genomic DNA of E. coli K12 and introduced into E. coli WT5. The expression of aroP and tyrR were detected and the activities of AroP and TyrR were determined. The introduction of either aroP(p) or aroP elevated the strain's transport activity by 1.40 or 1.46-fold respectively. Transformant carrying tyrR gene showed an ATPase activity 1.69-fold compared with the control. When the genes were linked in tandem and co-expressed in a plasmid, the relative AroP transport activity of the strain harboring aroP(p) -tyrR (0.95) was significantly lower than that of aroP-tyrR (1.31). The results indicated that TyrR might be able to reduce the expression of aroP gene by binding with the aroP promoter region in E.coli.

[Knockout of tyrR gene in Escherichia coli and its effects on the phenylalanine biosynthesis].

TyrR is a global regulation protein encoded by tyrR gene which controls several transcriptional units involving biosynthesis and transportation of aromatic amino acids in Escherichia coli. In this work, the tyrR gene was knocked out by a double-cross homologous recombination. The tyrR- mutant was verified by structural identification by PCR and sequencing, and functional demonstration using lacZ reporter gene. In tyrR- mutant, the activities of two key enzymes in the phenylalanine biosynthesis pathway, whose expression was controlled by TyrR, DAHPS and AT, had been shown to elevate by a 1.08-fold and 2.70-fold compared with the parent strain, respectively. The yield of phenylalanine biosynthesis in the mutant was 1.59 times higher than that of wild type strain. The repression on the transcription of aroP, encoding an aromatic amino acid permease, was eliminated, resulting in a 70.2% increase of the aromatic amino acid transportation in tyrR- mutant strain.

Co-expression of five genes in E coli for L-phenylalanine in Brevibacterium flavum.

AIM: To study the effect of co-expression of ppsA, pckA, aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strain for L-phenylalanine. METHODS: ppsA and pckA genes were amplified from genomic DNA of E. coli by polymerase chain reaction, and then introduced into shuttle vectors between E coli and Brevibacterium flavum to generate constructs pJN2 and pJN5. pJN2 was generated by inserting ppsA and pckA genes into vector pCZ; whereas pJN5 was obtained by introducing ppsA and pckA genes into pCZ-GAB, which was originally constructed for co-expression of aroG, pheA and tyrB genes. The recombinant plasmids were then introduced into B. flavum by electroporation and the transformants were used for L-phenylalanine fermentation. RESULTS: Compared with the original B. flavum cells, all the transformants were showed to have increased five enzyme activities specifically, and have enhanced L-phenylalanine biosynthesis ability variably. pJN5 transformant was observed to have the highest elevation of L-phenylalanine production by a 3.4-fold. Co-expression of ppsA and pckA increased activity of DAHP synthetase significantly. CONCLUSION: Co-expression of ppsA and pckA genes in B. flavum could remarkably increase the expression of DAHP synthetase; Co-expression of ppsA, pckA, aroG, pheA and tyrB of E. coli in B. flavum was a feasible approach to construct a strain for phenylalanine production.

Cloning and Expression of aroG Gene of E. coli and Its Co-expression with pheA and tyrB Genes.

3-Deoxy-D-arabino-heptulonate-7-phosphate synthetase (DAHP) is one of the key enzymes in phenylalanine biosynthesis pathway. In E. coli, DAHP is encoded by aroG Gene. In this work, aroG was cloned from an E. coli mutant strain resistant to m-fluro-L-phenylalanine (mPF) and p-fluro-L-phenylalanine (pPF) by PCR. The gene was expressed under the control of lambda phage promoter p(R) in P2392 strain of E. coli. Distinct band was detected as the product of aroG on SDS-PAGE. The specific activity in crude extract of DAHP was raised to 1.7-fold. Based on the cloning and expression of pheA (encoding both chorsmate mutase CM and prephenate dehydratase PD) and tyrB (encoding phenylalanine aminotransferase PAT) genes, aroG, pheA and tyrB genes were constructed and expressed in P2392. The results showed that the specific activities of DAPH, CM/PD and PAT in crude extracts were increased by 1.7, 13.9/7.8 and 2.3-fold, respectively.