Digital polymerase chain reaction ( PCR) has been experiencing a rapid development during the past few years. Comparing with the traditional real-time quantitative PCR ( RT-qPCR) , using the same primer and probe, the accuracy for the absolute quantification of target gene is significantly improved. The development of digital PCR is directly related to the development of microfluidics. The integrated fluid circuit is an early combination of the microfluidics and digital PCR, which has a complicated fabrication process with high cost. Recently, researchers are trying to apply the droplet microfluidics in digital PCR, and the droplet microfluidic chip is able to generate millions of droplets within a short time. Each of these droplets containing no more than one target gene is a reaction chamber during the amplification process. After amplification, each droplet is tested to achieve the absolute quantification of the target gene. This paper reviews the recent progresses of droplet digital PCR, and the applications of droplet digital PCR in biological, medical and environmental fields.

wearable microfluidics have wide applications in medical, sports and military field. with the help of wearable microfluidics, through the direct physical contact between the chip and skin, the pH, glucose, lactate, sodium/potassium, calcium and heavy metal in the body fluid can be detected from sweat, tear and saliva without puncture for blood. And these information are of great importance for the monitoring of vital signs and disease diagnosis. This paper introduced the most recent development and applications of the wearable microfluidics in the body fluid testing and drug delivery. The up-to-date research development for the drug delivery using wearable microfluidics was also briefly introduced in this article. The forecast of the emerging trend for wearable microfluidics and discussion of potential technique barriers was also provided at the end of this article.

Objective:To investigate high mobility group protein 1(HMGB1), tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)levels and their clinical significance in elderly patients with viral pneumonia.Methods:One hundred and sixty elderly patients with viral pneumonia admitted to the Sixth Hospital Affiliated to Anhui Medical University were enrolled as research subjects.In addition, 40 elderly people who underwent regular physical examination were considered as the control group.Patients with viral pneumonia were divided into the low-risk group, middle-risk group and high-risk group according to CURB-65 scores and pneumonia severity index(PSI)scores.HMGB1, TNF-α and IL-6 levels were compared between different groups.The correlations of CURB-65 scores and PSI scores with HMGB1, TNF-α, IL-6 levels were analyzed.Multivariate Logistic regression analysis was used to examine influencing factors for the severity of viral pneumonia in elderly patients.Results:HMGB1, TNF-α and IL-6 levels were higher in research subjects than in the control group.As the severity of viral pneumonia increased, so did HMGB1, TNF-α and IL-6 levels(all P<0.05). HMGB1, TNF-α and IL-6 levels in the severe viral pneumonia group were significantly higher than those in the non-severe viral pneumonia group( P<0.05). HMGB1, TNF-α and IL-6 levels were positively correlated with CURB-65 scores and PSI scores(CURB-65 score: r=0.463, 0.392 and 0.497, P=0.015, 0.003 and 0.025; PSI score: r=0.596, 0.515 and 0.381, P=0.007, 0.011 and 0.009). HMGB1, TNF-α and IL-6 levels were influencing factors for the severity of viral pneumonia in elderly patients( OR=1.344, 1.422 and 1.351, P=0.006, 0.015 and 0.009). Conclusions:HMGB1, TNF-α and IL-6 levels are closely correlated with the severity of viral pneumonia and are helpful for early assessment of viral pneumonia.

In the present study, we examined the regulation of the expression and function of AB CA1 by modified LDL (ox-LDL) in vitro. After incubation with apoA-Ⅰ for 24 h, RAW264.7 cells effluxed 37.65 % cholesterol loaded by acetyl LDL (ac-LDL), and 9.78 % cholesterol in ox-LDL group. The level of ABCA1 Mrna increased about three times either when cells were incubated with 100 μg/Ml ac-LDL or with 100μg/Ml ox-LDL. However, the level of ABCA1 protein rose by 1.57 times in ac-LDL group and 1.26 times in ox-LDL group. These results demonstrated that ox-LDL had different effect on the expression and function of ABCA1, ox-LDL might decrease the cholesterol efflux mediated by ABCA1 through other unknown mechanisms.

Objective:To establish an experimental model for intracellular antibacteria and endotoxin neutralization in vitro to detect the antibacterial and endotoxin neutralization activity of the muBPI_(25) protein.Methods: RAW264.7 cells were transfected with pcDNA3.1(+)muBPI_(36-259), and then were infected with intracellular bacterial of either G ~+/G~-to establish the experimental model of intracellalar antibacteria.The RAW264.7 cells were co-transfected with the pSecTag2B-muBPI_(36-259) and dual-luciferase reporter gene plasmids for establishment of the experimental model of endotoxin neutralization.Results:The experimental model of intracellular antibacteria confirmed that the muBPI_(25) protein could inhibit/kill Salmonella typhi.The experimental model of endotoxin neutralization indicated that the muBPI_(25) protein could neutralize endotoxin.Conelusion: We firstly demonstrate that murine BPI N-terminal functional fragment(muBPI_(25) protein)can inhibit/kill Salmonella typhi,and can neutralize, its lysating product, endotoxin.

To explore the intracellular signal pathways for beta-VLDL induced very low density lipoprotein receptor (VLDL-R) transcription up-regulation and their effects on lipid accumulation in macrophages, Western Blot was used to examine phosphorylated ERK1/2 protein and regulated effects by different singal kinase inhibitants. It was found that beta-VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. By using different protein kinases inhibitors or activators, it was observed that the effect of beta-VLDL induced VLDL receptor transcription, which was monitored by RT-PCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but extremely abolished by pretreating cells with PD98059, an inhibitor of ERK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC-ERK1/2 cascade is the essential signaling pathway by which beta-VLDL activated VLDL-R mRNA expression. Inhibition of the ERK1/2 signaling cascade resulted in suppression of the cellular lipid accumulation induced by beta-VLDL in macrophages.
Cells, Cultured ; Lipoproteins, VLDL ; metabolism ; Macrophages ; cytology ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; physiology ; Mitogen-Activated Protein Kinase 3 ; metabolism ; physiology ; Protein Kinase C ; antagonists & inhibitors ; metabolism ; Receptors, LDL ; biosynthesis ; genetics ; Signal Transduction ; Transcription Factors ; metabolism ; Transcription, Genetic ; Up-Regulation

To elucidate the intracellular signaling pathways for VLDL-induced VLDLR transcription, Western blot analysis was used to examine phosphorylated ERK1/2 protein. It was found that that VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. By using different protein kinases inhibitors or activators it was observed that the effect of VLDL-induced VLDL receptor transcription, which is monitored by RTPCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but completely abolished by pretreatment of the cells with PD 98059, an inhibitor of MEK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC/ERK1/2 cascade is the essential signaling pathway by which VLDL activates VLDL receptor mRNA expression.
Animals ; CCAAT-Binding Factor ; metabolism ; Cattle ; Cells, Cultured ; Gene Expression Regulation ; Humans ; Lipoproteins, VLDL ; metabolism ; Macrophages ; cytology ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases ; metabolism ; physiology ; Muscle, Smooth, Vascular ; metabolism ; Phosphorylation ; RNA, Messenger ; metabolism ; Rats ; Receptors, LDL ; metabolism ; Signal Transduction ; Transcription Factors ; metabolism ; Transcription, Genetic

Objective To explore the feasibility of 3D printed individualized applicator for the intracavitary HDR-brachytherapy for nasopharyngeal carcinoma.Methods CT scan was performed in 1 case of recurrent rT1 nasopharyngeal carcinoma and 1 case of T2 residual nasopharyngeal carcinoma and the obtained images were transmitted to 3D image processing software.The geometric contour parameters of the nasopharyngeal cavity were obtained and a pipeline was designed to make it close to the recurrent gross tumor volume (rGTV).Individualized cavity applicators were created by using 3D printer.The applicator was inserted into the patient's nasopharyngeal cavity through oral cavity.The source tube and false source were inserted into the preset pipe of the applicator.CT scan was performed again and the images were transmitted to the 3D brachytherapy planning system.Mter delineating the target volume and organ at risk,treatment plan was optimized.After completing the first treatment,the applicator was removed.Before second treatment in a few days,CT scan was reviewed to confirm whether the position was correct.Results When the applicator was inserted into the nasopharyngeal cavity,it could be fully aligned with the nasopharyngeal wall and self-fixed without additional fixation measures.Comparing the location of false source in multiple reviews of CT scan,the error was ≤ 1 mm.No significant discomfort was reported throughout the treatment.In optimized three-dimensional treatment,100％ prescription dose curve included the full rGTV,maximum dose of the brain stem and spinal cord was＜30％ prescription dose.Recurrent patients were given with a prescription dose of DT 40Gy/8 fractions/4 weeks and patients with residual tumors were given with 12Gy/2 fractions/1 week.No tumor recurrence was observed at postoperative 3 months in two cases.Conclusions The 3D printed individualized nasopharyngeal intracavitary applicator has the advantages of self-fixation,accurate location,good repeatability and good patient tolerance.The short-term outcome is effective,whereas its long-term clinical effect and adverse reactions need to be further observed.

The role of very low density lipoprotein receptor (LVLDR) in the process of foam cell formation was investigated. After the primary cultured mouse peritoneal macrophages were incubated with VLDL, beta-VLDL or low density lipoprotein (LDL), respectively for 24 h and 48 h, foam cells formation was identified by oil red O staining and cellular contents of triglyceride (TG) and total cholesterol (TC) were determined. The mRNA levels of LDLR, LDLR related protein (ILRP) and VLDLR were detected by semi-quantitative RT-PCR. The results demonstrated that VLDL, beta-VLDL and LDL could increase the contents of TG and TC in macrophages. Cells treated with VLDL or beta-VLDL showed markedly increased expression of VLDLR and decreased expression of LDLR, whereas LRP was up-regulated slightly. For identifying the effect of VLDL receptor on cellular lipid accumulation, ldl-A7-VR cells, which expresses VLDLR and trace amount of LRP without functional LDLR, was used to incubate with lipoproteins for further examination. The results elucidated that the uptake of triglyceride-rich lipoprotein mediated by VLDLR plays an important role in accumulation of lipid and the formation of foam cells.
Animals ; Arteriosclerosis ; metabolism ; pathology ; Cells, Cultured ; Cholesterol, LDL ; metabolism ; pharmacology ; Female ; Foam Cells ; cytology ; metabolism ; Lipoproteins, VLDL ; pharmacology ; Macrophages, Peritoneal ; cytology ; metabolism ; Mice ; Receptors, LDL ; metabolism ; Triglycerides ; metabolism