Studies of Antibiotic Resistance of Beta-Lactamase Bacteria under Different Nutrition Limitations at the Single-Cell Level

Drug resistance involves many biological processes, including cell growth, cell communication, and cell cooperation. In the last few decades, bacterial drug resistance studies have made substantial progress. However, a major limitation of the traditional resistance study still exists: most of the studies have concentrated on the average behavior of enormous amounts of cells rather than surveying single cells with different phenotypes or genotypes. Here, we report our study of beta-lactamase bacterial drug resistance in a well-designed microfluidic device, which allows us to conduct more controllable experiments, such as controlling the nutrient concentration, switching the culture media, performing parallel experiments, observing single cells, and acquiring time-lapse images. By using GFP as a beta-lactamase indicator and acquiring time-lapse images at the single-cell level, we observed correlations between the bacterial heterogeneous phenotypes and their behavior in different culture media. The feedback loop between the growth rate and the beta-lactamase production suggests that the beta-lactamase bacteria are more resistant in a rich medium than in a relatively poor medium. In the poorest medium, the proportion of dormant cells may increase, which causes a lower death rate in the same generation. Our work may contribute to assaying the antibiotic resistance of pathogenic bacteria in heterogeneous complex media.     

Transforming Growth Factor TGFβ Increases Levels of Microtubule-Associated Protein MAP1S and Autophagy Flux in Pancreatic Ductal Adenocarcinomas

Background and Aim

Autophagy is a cellular process to regulate the turnover of misfolded/aggregated proteins or dysfunctional organelles such as damaged mitochondria. Microtubule-associated protein MAP1S (originally named C19ORF5) is a widely-distributed homologue of neuronal-specific MAP1A and MAP1B with which autophagy marker light chain 3 (LC3) was originally co-purified. MAP1S bridges autophagic components with microtubules and mitochondria through LC3 and positively regulates autophagy flux from autophagosomal biogenesis to degradation. The MAP1S-mediated autophagy suppresses tumorigenesis as suggested in a mouse liver cancer model and in prostate cancer patients. The TGFβ signaling pathway plays a central role in pancreatic tumorigenesis, and high levels of TGFβ suggest a tumor suppressive function and predict a better survival for some patients with resectable pancreatic ductal adenocarcinoma. In this study, we try to understand the relationship between TGFβ and MAP1S-mediated autophagy in pancreatic ductal adenocarcinoma.

Methods

We collected the tumor and its adjacent normal tissues from 33 randomly selected patients of pancreatic ductal adenocarcinomas to test the association between TGFβ and autophagy markers MAP1S and LC3. Then we tested the cause and effect relation between TGFβ and autophagy markers in cultured pancreatic cancer cell lines.

Results

Here we show that levels of TGFβ and autophagy markers MAP1S and LC3 are dramatically elevated in tumor tissues from patients with pancreatic ductal adenocarcinomas. TGFβ increases levels of MAP1S protein and enhances autophagy flux.

Conclusion

TGFβ may suppress the development of pancreatic ductal adenocarcinomas by enhancing MAP1S-mediated autophagy.     

Impaired Function of CD5+CD19+CD1dhi B10 Cells on IgE Secretion in an Atopic Dermatitis-Like Mouse Model

Atopic dermatitis (AD) is a chronic inflammatory pruritic skin disease in which the pathogenic mechanism is complicated and not completely understood. Reports on the role of regulated cells in AD have recently evolved to regulate B cells, which may play a role in allergic inflammation as well. In the present study, we examined the frequency and regulatory function of CD5⁺CD19⁺CD1d^hi B10 cells in an AD-like mouse model. Our results showed that the percentage of CD5⁺CD19⁺CD1d^hi B10 cells increased while the frequency of IL-10-producing B cells in CD19⁺B cells decreased in the mice of AD group. Moreover, no difference in the percentage of B10pro+B10 cells was observed between the AD and control groups. Strikingly, B10 cells from control mice effectively inhibited IgE secretion, whereas the suppressive function of B10 cells from the AD mice was significantly decreased, which was similar to that observed in the group without B10. Altogether, these results suggest that the number of IL-10-producing B cells decreased in the AD group and these cells showed a defective regulatory function on IgE secretion.     

Trans-Corneal Subretinal Injection in Mice and Its Effect on the Function and Morphology of the Retina

Purpose

To introduce a practical method of subretinal injection in mice and evaluate injection-induced retinal detachment (RD) and damage using a dynamic imaging system, electrophysiology, and histology.

Methods

After full dilation of a 2-month-old C57BL/6J mouse pupil, the cornea near the limbus was punctured with a 30 ½-gague disposable beveled needle. A 33 ½-gauge blunt needle was inserted through the corneal perforation into the anterior chamber, avoiding the lens before going deeper into the vitreous cavity, and penetrating the inner retina to reach the subretinal space. The mice were divided into four groups: in group 1, about 80–100% of the retina was filled with subretinally injected solution; in group 2, approximately 50–70% of the retina was filled with injected solution; in group 3, the procedures were stopped before solution injection; and non-injected eyes were used as the negative control in group 4. An optical coherence tomography (OCT) imaging system was used to monitor retinal reattachment during the first three days following the injections. Histological and functional changes were examined by light microscopy and electroretinography (ERG) at five weeks post-injection.

Results

After a short-term training, a 70% success rate with 50% or more coverage (i.e., retinal blebs occupied 50% or more retinal area and filled with the injected solution) with minimal injection-related damages can be achieved. Bleb formation was associated with retinal detachment (RD) between the neuroretina and the retinal pigment epithelium (RPE) layer. Partial RD could be observed at post-injection day 1, and by day 2 most of the retina had reattached. At 5 weeks post-injection, compared to uninjected control group 4, the b-wave amplitudes of ERG decreased 22% in group 1, 16% in group 2, and 7% in group 3; the b-wave amplitudes were statistically different between the uninjected group and the groups with either 50–70% or 80–100% coverage. The subretinal injection-induced RD reattached and became stable at five weeks post-injection, although some photoreceptor damage could still be observed in and around the injection sites, especially in 80–100% coverage group.

Conclusions

Trans-corneal subretinal injection is effective and practical, although subretinal injection-related damages can cause some morphological and functional loss.     

Cadmium-Induced Hydrogen Accumulation Is Involved in Cadmium Tolerance in Brassica campestris by Reestablishment of Reduced Glutathione Homeostasis

Hydrogen gas (H₂) was recently proposed as a therapeutic antioxidant and signaling molecule in clinical trials. However, the underlying physiological roles of H₂ in plants remain unclear. In the present study, hydrogen-rich water (HRW) was used to characterize the physiological roles of H₂ in enhancing the tolerance of Brassica campestris against cadmium (Cd). The results showed that both 50 μM CdCl₂ and 50%-saturated HRW induced an increase of endogenous H₂ in Brassica campestris seedlings, and HRW alleviated Cd toxicity related to growth inhibition and oxidative damage. Seedlings supplied with HRW exhibited increased root length and reduced lipid peroxidation, similar to plants receiving GSH post-treatment. Additionally, seedlings post-treated with HRW accumulated higher levels of reduced glutathione (GSH) and ascorbic acid (AsA) and showed increased GST and GPX activities in roots. Molecular evidence illustrated that the expression of genes such as GS, GR1 and GR2, which were down-regulated following the addition of Cd, GSH or BSO, could be reversed to varying degrees by the addition of HRW. Based on these results, it could be proposed that H₂ might be an important regulator for enhancing the tolerance of Brassica campestris seedlings against Cd, mainly by governing reduced glutathione homeostasis.     

Warmer and Wetter Soil Stimulates Assimilation More than Respiration in Rainfed Agricultural Ecosystem on the China Loess Plateau: The Role of Partial Plastic Film Mulching Tillage

Effects of agricultural practices on ecosystem carbon storage have acquired widespread concern due to its alleviation of rising atmospheric CO₂ concentrations. Recently, combining of furrow-ridge with plastic film mulching in spring maize ecosystem was widely applied to boost crop water productivity in the semiarid regions of China. However, there is still limited information about the potentials for increased ecosystem carbon storage of this tillage method. The objective of this study was to quantify and contrast net carbon dioxide exchange, biomass accumulation and carbon budgets of maize (Zea maize L.) fields under the traditional non-mulching with flat tillage (CK) and partial plastic film mulching with furrow-ridge tillage (MFR) on the China Loess Plateau. Half-hourly net ecosystem CO₂ exchange (NEE) of both treatments were synchronously measured with two eddy covariance systems during the growing seasons of 2011 through 2013. At same time green leaf area index (GLAI) and biomass were also measured biweekly. Compared with CK, the warmer and wetter (+1.3°C and +4.3%) top soil at MFR accelerated the rates of biomass accumulation, promoted greater green leaf area and thus shortened the growing seasons by an average value of 10.4 days for three years. MFR stimulated assimilation more than respiration during whole growing season, resulting in a higher carbon sequestration in terms of NEE of -79 gC/m² than CK. However, after considering carbon in harvested grain (or aboveground biomass), there is a slight higher carbon sink (or a stronger carbon source) in MFR due to its greater difference of aboveground biomass than that of grain between both treatments. These results demonstrate that partial plastic film mulched furrow-ridge tillage with aboveground biomass exclusive of grain returned to the soil is an effective way to enhance simultaneously carbon sequestration and grain yield of maize in the semiarid regions.     

One-step homogeneous non-stripping chemiluminescence metal immunoassay based on catalytic activity of gold nanoparticles

The catalytic activity of gold nanoparticles (AuNPs) on a luminol–H₂O₂ chemiluminescence (CL) system is found to be greatly enhanced after its crosslinking aggregation induced by immunoreaction. Based on this observation, a one-step homogeneous non-stripping CL metalloimmunoassay was designed. In the presence of corresponding antigen (Ag), the immunoreaction caused the aggregation of antibody (Ab)-modified AuNPs, and these crosslinking aggregated AuNPs could catalyze luminol–H₂O₂ CL reaction to produce a much stronger CL signal than dispersed Ab-modified AuNPs. The assay, including immunoreaction and detection, can be accomplished in homogeneous solution. In the assay, no tedious and strict stripping of metal nanoparticles, difficult synthesis of labels, multiple steps of immunoreactions and washings, and complicated magnetic separation process were required. The detection limit of human immunoglobulin G (IgG, 3σ) was estimated to be as low as 3.2 × 10⁻¹¹ g ml⁻¹. The sensitivity was increased by two orders of magnitude over that of other AuNP-based CL immunoassay. The current CL metalloimmunoassay offers the advantages of being simple, cheap, rapid, and sensitive.     

miR-329 suppresses the growth and motility of neuroblastoma by targeting KDM1A

MicroRNAs (miRNAs) act as key regulators of multiple cancers. miR-329 functions as a tumor suppressor in some malignancies. However, its role in neuroblastoma remains poorly understood. We found that miR-329 was decreased in metastatic tumor tissues compared with matched primary tumor tissues. Forced overexpression of miR-329 substantially suppressed cell proliferation, colony formation, migration, and invasion of neuroblastoma cells. Lysine-specific demethylase 1 (KDM1A) was found to be a target of miR-329. Furthermore, down-regulation of KDM1A by shRNA performed similar effects with overexpression of miR-329. Overexpression of KDM1A partially reversed the tumor suppressive effects of miR-329 in neuroblastoma cells. Collectively, miR-329 may suppress neuroblastoma cell growth and motility partially by targeting KDM1A.