This work describes fully automated and colorimetric foodborne pathogen detection on an integrated centrifugal microfluidic device, which is called a lab-on-a-disc. All the processes for molecular ...diagnostics including DNA extraction and purification, DNA amplification and amplicon detection were integrated on a single disc. Silica microbeads incorporated in the disc enabled extraction and purification of bacterial genomic DNA from bacteria-contaminated milk samples. We targeted four kinds of foodborne pathogens (Escherichia coli O157:H7, Salmonella typhimurium, Vibrio parahaemolyticus and Listeria monocytogenes) and performed loop-mediated isothermal amplification (LAMP) to amplify the specific genes of the targets. Colorimetric detection mediated by a metal indicator confirmed the results of the LAMP reactions with the colour change of the LAMP mixtures from purple to sky blue. The whole process was conducted in an automated manner using the lab-on-a-disc and a miniaturized rotary instrument equipped with three heating blocks. We demonstrated that a milk sample contaminated with foodborne pathogens can be automatically analysed on the centrifugal disc even at the 10 bacterial cell level in 65 min. The simplicity and portability of the proposed microdevice would provide an advanced platform for point-of-care diagnostics of foodborne pathogens, where prompt confirmation of food quality is needed.
It remains controversial whether targeting tumour vasculature can improve radiotherapeutic efficacy. We report that radiation-induced endothelial-to-mesenchymal transition (EndMT) leads to tumour ...vasculature with abnormal SMA
NG2
pericyte recruitment during tumour regrowth after radiotherapy. Trp53 (but not Tgfbr2) deletion in endothelial cells (ECs) inhibited radiation-induced EndMT, reducing tumour regrowth and metastases with a high CD44v6
cancer-stem-cell (CSC) content after radiotherapy. Osteopontin, an EndMT-related angiocrine factor suppressed by EC-Trp53 deletion, stimulated proliferation in dormant CD44v6
cells in severely hypoxic regions after radiation. Radiation-induced EndMT significantly regulated tumour-associated macrophage (TAM) polarization. CXCR4 upregulation in radioresistant tumour ECs was highly associated with SDF-1
TAM recruitment and M2 polarization of TAMs, which was suppressed by Trp53 deletion. These EndMT-related phenomena were also observed in irradiated human lung cancer tissues. Our findings suggest that targeting tumour EndMT might enhance radiotherapy efficacy by inhibiting the re-activation of dormant hypoxic CSCs and promoting anti-tumour immune responses.
Rectal cancer (RC) is a challenging disease to treat that requires chemotherapy, radiation and surgery to optimize outcomes for individual patients. No accurate model of RC exists to answer ...fundamental research questions relevant to patients. We established a biorepository of 65 patient-derived RC organoid cultures (tumoroids) from patients with primary, metastatic or recurrent disease. RC tumoroids retained molecular features of the tumors from which they were derived, and their ex vivo responses to clinically relevant chemotherapy and radiation treatment correlated with the clinical responses noted in individual patients' tumors. Upon engraftment into murine rectal mucosa, human RC tumoroids gave rise to invasive RC followed by metastasis to lung and liver. Importantly, engrafted tumors displayed the heterogenous sensitivity to chemotherapy observed clinically. Thus, the biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based, ex vivo platform coupled with in vivo endoluminal propagation in animals.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Endothelial-to-mesenchymal transition (EndMT) involves the phenotypic conversion of endothelial-to-mesenchymal cells, and was first discovered in association with embryonic heart development. EndMT ...can regulate various processes, such as tissue fibrosis and cancer. Recent findings have shown that EndMT is related to resistance to cancer therapy, such as chemotherapy, antiangiogenic therapy, and radiation therapy. Based on the known effects of EndMT on the cardiac toxicity of anticancer therapy and tissue damage of radiation therapy, we propose that EndMT can be targeted as a strategy for overcoming tumor resistance while reducing complications, such as tissue damage. In this review, we discuss EndMT and its roles in damaging cardiac and lung tissues, as well as EndMT-related effects on tumor vasculature and resistance in anticancer therapy. Modulating EndMT in radioresistant tumors and radiation-induced tissue fibrosis can especially increase the efficacy of radiation therapy. In addition, we review the role of hypoxia and reactive oxygen species as the main stimulating factors of tissue damage due to vascular damage and EndMT. We consider drugs that may be clinically useful for regulating EndMT in various diseases. Finally, we argue the importance of EndMT as a therapeutic target in anticancer therapy for reducing tissue damage.
In this study, we developed a centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria contaminated milk ...samples. The microdevice was designed to contain identical triplicate functional units and each unit has four reaction chambers, thereby making it possible to perform twelve direct-RPA reactions simultaneously. The integrated microdevice consisted of two layers: RPA reagents were injected in the top layer, while spiked milk samples with food poisoning bacteria were loaded into sample reservoirs in the bottom layer. For multiplex bacterial detection, the target gene-specific primers and probes were dried in each reaction chamber. The introduced samples and reagents could be equally aliquoted and dispensed into each reaction chamber by centrifugal force, and then the multiplex direct-RPA reaction was executed. The target genes of bacteria spiked in milk could be amplified at 39 °C without a DNA extraction step by using the direct-RPA cocktails, which were a combination of a direct PCR buffer and RPA enzymes. As the target gene amplification proceeded, the increased fluorescence signals coming from the reaction chambers were recorded in real-time at an interval of 2 min. The entire process, including the sample distribution, the direct-RPA reaction, and the real-time analysis, was accomplished with a custom-made portable genetic analyzer and a miniaturized optical detector. Monoplex, duplex, and triplex food poisoning bacteria (Salmonella enterica, Escherichia coli O157:H7, and Vibrio parahaemolyticus) detection was successfully performed with a detection sensitivity of 4 cells per 3.2 μL of milk samples within 30 min. By implementing the direct-PRA on the miniaturized centrifugal microsystem, the on-site food poisoning bacteria analysis would be feasible with high speed, sensitivity, and multiplicity.
Targeting the molecular pathways underlying the cardiotoxicity associated with thoracic irradiation and doxorubicin (Dox) could reduce the morbidity and mortality associated with these anticancer ...treatments. Here, we find that vascular endothelial cells (ECs) with persistent DNA damage induced by irradiation and Dox treatment exhibit a fibrotic phenotype (endothelial-mesenchymal transition, EndMT) correlating with the colocalization of L1CAM and persistent DNA damage foci. We demonstrate that treatment with the anti-L1CAM antibody Ab417 decreases L1CAM overexpression and nuclear translocation and persistent DNA damage foci. We show that in whole-heart-irradiated mice, EC-specific p53 deletion increases vascular fibrosis and the colocalization of L1CAM and DNA damage foci, while Ab417 attenuates these effects. We also demonstrate that Ab417 prevents cardiac dysfunction-related decrease in fractional shortening and prolongs survival after whole-heart irradiation or Dox treatment. We show that cardiomyopathy patient-derived cardiovascular ECs with persistent DNA damage show upregulated L1CAM and EndMT, indicating clinical applicability of Ab417. We conclude that controlling vascular DNA damage by inhibiting nuclear L1CAM translocation might effectively prevent anticancer therapy-associated cardiotoxicity.
Zbtb7c is a proto-oncoprotein that controls the cell cycle and glucose, glutamate, and lipid metabolism. Zbtb7c expression is increased in the liver and white adipose tissues of aging or high-fat ...diet-fed mice. Knockout or knockdown of Zbtb7c gene expression inhibits the adipocyte differentiation of 3T3-L1 cells and decreases adipose tissue mass in aging mice. We found that Zbtb7c was a potent transcriptional repressor of SIRT1 and that SIRT1 was derepressed in various tissues of Zbtb7c-KO mice. Mechanistically, Zbtb7c interacted with p53 and bound to the proximal promoter p53RE1 and p53RE2 to repress the SIRT1 gene, in which p53RE2 was particularly critical. Zbtb7c induced p53 to interact with the corepressor mSin3A-HADC1 complex at p53RE. By repressing the SIRT1 gene, Zbtb7c increased the acetylation of Pgc-1α and Pparγ, which resulted in repression or activation of Pgc-1α or Pparγ target genes involved in lipid metabolism. Our study provides a molecular target that can overexpress SIRT1 protein in the liver, pancreas, and adipose tissues, which can be beneficial in the treatment of diabetes, obesity, longevity, etc.
Adhesion is a primary challenge following surgery, and the anti-adhesive effect of methylene blue (MB) has been investigated. This systematic review and meta-analysis aimed to evaluate the effect of ...MB on postoperative adhesions in experimental studies. We initially searched OVID-MEDLINE, EMBASE, and Google Scholar in February 2021, and then in May 2021. The anti-adhesive efficacy of MB was compared with that of the control (either placebo or nothing) after the surgical procedure. The primary and secondary outcomes were the macroscopic and microscopic adhesion scores, respectively. Traditional meta-analysis, meta-regression, and trial sequential analysis (TSA) were performed to analyze the retrieved outcomes. We included 13 experimental studies of 367 rats (200 rats received MB and 167 rats received placebo or nothing). The macroscopic adhesion scores were significantly lower in the MB-administered group than in the control group (standardized mean difference, 2.313; 95% confidence interval, 1.104 to3.523; I2 = 94.0%, Tau = 2.059). Meta-regression analysis showed that macroscopic adhesion tended to decrease with an increase in MB dose. TSA demonstrated that the cumulative Z curve crossed both the conventional test and trial sequential monitoring boundary for the macroscopic adhesion score. MB had a beneficial effect on intraperitoneal adhesion following laparotomy, and adhesions decreased with increase in dose.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Brown adipose tissue generates heat via the mitochondrial uncoupling protein UCP1 to protect against obesity and hypothermia. Fas mutant MRL/lpr mice exhibit a significantly leaner phenotype compared ...to wild type MRL/MpJ mice. In this study, we evaluated the inflammatory cell population in the adipose tissue of MRL/lpr mice, which could potentially influence their lean phenotype. Furthermore, we compared beige fat activity between the MRL/MpJ and MRL/lpr mice. Fas mutation resulted in high body temperature, improved glucose tolerance, and decreased fat mass and adipocyte size. Fas mutation prevented high-fat diet-induced obesity and decreased the white adipose tissue M1:M2 ratio. When mice were fed a high-fat diet, UCP1, IL-4, IL-10, and tyrosine hydroxylase genes had significantly higher expression in Fas-mutant mice than in wild type mice. After a cold challenge, UCP1 expression and browning were also significantly higher in the Fas-mutant mice. In summary, Fas-mutant mice are resistant to high-fat diet-induced obesity due to increased IL-4 and IL-10 levels and the promotion of thermogenic protein activity and browning in their adipose tissues. STAT6 activation might contribute to M2 polarisation by increasing IL-4 and IL-10 levels while increases in M2 and tyrosine hydroxylase levels promote browning in response to Fas mutation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
3‐Fucosyllactose (3‐FL) is one of the major fucosylated oligosaccharides in human milk. Along with 2′‐fucosyllactose (2′‐FL), it is known for its prebiotic, immunomodulator, neonatal brain ...development, and antimicrobial function. Whereas the biological production of 2′‐FL has been widely studied and made significant progress over the years, the biological production of 3‐FL has been hampered by the low activity and insoluble expression of α‐1,3‐fucosyltransferase (FutA), relatively low abundance in human milk oligosaccharides compared with 2′‐FL, and lower digestibility of 3‐FL than 2′‐FL by bifidobacteria. In this study, we report the gram‐scale production of 3‐FL using E. coli BL21(DE3). We previously generated the FutA quadruple mutant (mFutA) with four site mutations at S46F, A128N, H129E, Y132I, and its specific activity was increased by nearly 15 times compared with that of wild‐type FutA owing to the increase in kcat and the decrease in Km. We overexpressed mFutA in its maximum expression level, which was achieved by the optimization of yeast extract concentration in culture media. We also overexpressed L‐fucokinase/GDP‐
L‐fucose pyrophosphorylase to increase the supply of GDP‐fucose in the cytoplasm. To increase the mass of recombinant whole‐cell catalysts, the host E. coli BW25113 was switched to E. coli BL21(DE3) because of the lower acetate accumulation of E. coli BL21(DE3) than that of E. coli BW25113. Finally, the lactose operon was modified by partially deleting the sequence of LacZ (lacZΔm15) for better utilization of D‐lactose. Production using the lacZΔm15 mutant yielded 3‐FL concentration of 4.6 g/L with the productivity of 0.076 g·L−1·hr−1 and the specific 3‐FL yield of 0.5 g/g dry cell weight.
3‐Fucosyllactose (3‐FL) is one of the major fucosylated oligosaccharides in human milk. Along with 2′‐fucosyllactose (2′‐FL), it is known for its prebiotic, immunomodulator, neonatal brain development, and antimicrobial function.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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