Activationless Electron Transfer of Redox-DNA in Electrochemical Nanogaps.

Our recent discovery of decreased reorganization energy in electrode-tethered redox-DNA systems prompts inquiries into the origin of this phenomenon and suggests its potential use to lower the activation energy of electrochemical reactions. Here, we show that the confinement of the DNA chain in a nanogap amplifies this effect to an extent to which it nearly abolishes the intrinsic activation energy of electron transfer. Employing electrochemical atomic force microscopy (AFM-SECM), we create sub-10 nm nanogaps between a planar electrode surface bearing end-anchored ferrocenylated DNA chains and an incoming microelectrode tip. The redox cycling of the DNA's ferrocenyl (Fc) moiety between the surface and the tip generates a measurable current at the scale of ∼10 molecules. Our experimental findings are rigorously interpreted through theoretical modeling and original molecular dynamics simulations (Q-Biol code). Several intriguing findings emerge from our investigation: (i) The electron transport resulting from DNA dynamics is many times faster than predicted by simple diffusion considerations. (ii) The current in the nanogap is solely governed by the electron transfer rate at the electrodes. (iii) This rate rapidly saturates as overpotentials applied to the nanogap electrodes increase, implying near-complete suppression of the reorganization energy for the oxidation/reduction of the Fc heads within confined DNA. Furthermore, evidence is presented that this may constitute a general, previously unforeseen, behavior of redox polymer chains in electrochemical nanogaps.

Electrochemical Shot Noise of a Redox Monolayer.

Redox monolayers are the base for a wide variety of devices including high-frequency molecular diodes or biomolecular sensors. We introduce a formalism to describe the electrochemical shot noise of such a monolayer, confirmed experimentally at room temperature in liquid. The proposed method, carried out at equilibrium, avoids parasitic capacitance, increases the sensitivity, and allows us to obtain quantitative information such as the electronic coupling (or standard electron transfer rates), its dispersion, and the number of molecules. Unlike in solid-state physics, the homogeneity in energy levels and transfer rates in the monolayer yields a Lorentzian spectrum. This first step for shot noise studies in molecular electrochemical systems opens perspectives for quantum transport studies in a liquid environment at room temperature as well as highly sensitive measurements for bioelectrochemical sensors.

Transcriptomic and proteomic studies suggest the establishment of advanced zonation-like profiles in human-induced pluripotent stem cell-derived liver sinusoidal endothelial cells and carboxypeptidase M-positive liver progenitor cells cocultured in a fluidic microenvironment.

AIM: Hepatic zonation is a physiological feature of the liver, known to be key in the regulation of the metabolism of nutrients and xenobiotics and the biotransformation of numerous substances. However, the reproduction of this phenomenon remains challenging in vitro as only part of the processes involved in the orchestration and maintenance of zonation are fully understood. The recent advances in organ-on-chip technologies, which allow for the integration of multicellular 3D tissues in a dynamic microenvironment, could offer solutions for the reproduction of zonation within a single culture vessel. METHODS: An in-depth analysis of zonation-related mechanisms observed during the coculture of human-induced pluripotent stem cell (hiPSC)-derived carboxypeptidase M-positive liver progenitor cells and hiPSC-derived liver sinusoidal endothelial cells within a microfluidic biochip was carried out. RESULTS: Hepatic phenotypes were confirmed in terms of albumin secretion, glycogen storage, CYP450 activity, and expression of specific endothelial markers such as PECAM1, RAB5A, and CD109. Further characterization of the patterns observed in the comparison of the transcription factor motif activities, the transcriptomic signature, and the proteomic profile expressed at the inlet and the outlet of the microfluidic biochip confirmed the presence of zonation-like phenomena within the biochips. In particular, differences related to Wnt/ß-catenin, transforming growth factor-ß, mammalian target of rapamycin, hypoxia-inducible factor-1, and AMP-activated protein kinase signaling, to the metabolism of lipids, and cellular remolding were observed. CONCLUSIONS: The present study shows the interest in combining cocultures of hiPSC-derived cellular models and microfluidic technologies for reproducing in vitro complex mechanisms such as liver zonation and further incites the use of those solutions for accurate reproduction of in vivo situations.

First report of leaf anthracnose caused by Colletotrichum camelliae on tea plants (Camellia sinensis) in South Korea.

The tea plant (Camellia sinensis (L.) O. Kuntze) is a popular non-alcoholic beverage crop worldwide. The tea market in South Korea is projected to increase annually by 4.59% (Statista, 2022). Boseong, Hadong, and Jeju Island are the main tea-growing regions in South Korea. Anthracnose is one of the major diseases of tea plants and is responsible for substantial yield loss and poor tea quality. In 2021, anthracnose of tea (disease incidence of 30%) was observed in a garden (33°28'45.5"N 126°42'02.2"E) at Jeju Island, where the Yabukita cultivar has been cultivated. The typical symptoms consisted of round or irregularly shaped lesions with gray-white centers and purple-brown borders. Twelve morphologically similar isolates were recovered from 12 infected leaves using the single spore isolation method on solid potato dextrose agar (PDA) (Cai et al. 2009). Four representative isolates (GT6, GT7, GT8, and GT11) were identified based on morphology, molecular analysis, and pathogenicity tests. The upper side of seven-day-old colonies on PDA (incubated at 25 °C in the dark) was off-white with white aerial mycelia and gray-white with black zonation on their reverse side. Conidia were hyaline, aseptate, cylindrical, with both obtuse ends, and measuring 12.3 - 25.8 µm × 4.4 - 9.3 µm (n = 50). Appressoria were dark brown, irregularly shaped with a smooth edge, and measuring 7.3 -18.8 µm × 6.9 - 11.3 µm (n = 50). According to morphological characteristics, the fungal isolates were tentatively identified as the Colletotrichum gloeosporioides complex, including C. caelliae (Wang et al. 2016; Weir et al. 2012). The genomic DNA was extracted, and the ribosomal internal transcribed spacer (ITS), ß-tubulin-2 (TUB2) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, actin (ACT), calmodulin (CAL), and the Apn2-Mat1-2 intergenic spacer and partial mating type (ApMat) genes were amplified and subsequently sequenced using primer sets ITS1/ITS4, BT2a/BT2b, GDF1/GDR1, ACT-512F/ACT-783R1, CL1C/CL2C, and AM-F/AM-R, respectively (Silva et al. 2012; Weir et al. 2012). The resulting sequences were deposited in GenBank accession numbers (LC738932-LC738959). All the representative isolates were identified as C. camelliae by constructing the 50% majority rule consensus and maximum likelihood phylogenetic treebased on the combined ITS, TUB2, GAPDH, ACT, CAL, and ApMat sequences using MrBayes v. 3.2.2 and Mega X, respectively (Kumar et al., 2018; Ronquist et al. 2012). The pathogenicity of these isolates was tested on healthy leaves of 2- years-old tea seedlings (the Yabukita cultivar). Onside of unwounded or wounded leaves of seedlings were inoculated with 20 µL of conidial suspension (1 × 106 conidia or spores/ml) per spot (3-4 wounded or unwounded spots per side per leaf). Another side of the leaves received sterile distilled water and served as a control. Each treatment was replicated three times (three seedlings/isolate and four leaves per seedling) and this experiment was repeated twice. All plants were covered with plastic bags, placed in a growth chamber, and incubated at 25 °C with a 12-h photoperiod and 90% relative humidity. Typical anthracnose symptoms appeared on wounded leaves after two days of inoculation. Unwounded and controlled leaves remain asymptotic. To confirm Koch's postulates, fungal isolates were re-isolated from inoculated leaf lesions and identified as C. camelliae based on morphology and ITS sequences. Colletotrichum camelliae is a very common pathogen associated with tea anthracnose worldwide, including China (Liu et al. 2015; Wang et al. 2016).To the best of our knowledge, this is the first report of anthracnose in tea trees caused by C. camelliae in South Korea. The results of this study could help come up with better ways to keep an eye on and deal with this devastating on tea plants. Key words: Tea anthracnose, Colletotrichum camelliae, pathogenicity References Cai, L., et al. 2009. Fungal Divers. 39:183. Kumar, S., et al. 2018. Mol. Biol. Evol. 35:1547. Liu, F. et al. 2015. Persoonia. 35: 63-86. Ronquist, F. et al. 2012. Syst. Biol. 61:539-542. Silva, D. N. et al. 2012. Mycologia. 104:396-409. Statista 2022. Statista Digital Market out Look. Available at www.statista.com. Wang, Y.-C. et al. 2016. Sci. Rep. 6: 35287. Weir, B. S., et al. 2012. Stud. Mycol. 73:115.

Direct Capture and Amplification of Small Fragmented DNAs Using Nitrogen-Mustard-Coated Microbeads.

Circulating cell-free DNA (cfDNA) has been implicated as an important biomarker and has been intensively studied for "liquid biopsy" applications in cancer diagnostics. Owing to its small fragment size and its low concentration in circulation, cfDNA extraction and purification from serum samples are complicated, and the extraction yield affects the precision of subsequent molecular diagnostic tests. Here, we report a novel approach using nitrogen-mustard-coated DNA capture beads (NMD beads) that covalently capture DNA and allow direct subsequent polymerase chain reaction (PCR) amplification from the NMD bead without elusion. The complex DNA extraction and purification processes are not required. To illustrate the diagnostic use of the NMD beads, we detected short DNA fragments (142 bp) that were spiked into fetal bovine serum (as a model serum sample). The spiked DNAs were captured directly from serum samples and detected using real-time PCR at concentrations as low as 10 fg/mL. We anticipate that this DNA capture bead technique has the potential to simplify the preanalytical processes required for cfDNA detection, which could significantly expand the diagnostic applications of liquid biopsy.

High-throughput sorting of nanoliter droplets enabled by a sequentially addressable dielectrophoretic array.

Droplet microfluidics has emerged as a powerful tool for a diverse range of biomedical and industrial applications such as single-cell analysis, directed evolution, and metabolic engineering. In these applications, droplet sorting has been effective for isolating small droplets encapsulating molecules, cells, or crystals of interest. Recently, there is an increased interest in extending the applicability of droplet sorting to larger droplets to utilize their size advantage. However, sorting throughputs of large droplets have been limited, hampering their wide adoption. Here, we report our demonstration of high-throughput fluorescence-activated droplet sorting of 1 nL droplets using an upgraded version of the sequentially addressable dielectrophoretic array (SADA), which we reported previously. The SADA is an array of electrodes that are individually and sequentially activated/deactivated according to the speed and position of a droplet passing nearby the array. We upgraded the SADA by increasing the number of driving electrodes constituting the SADA and incorporating a slanted microchannel. By using a ten-electrode SADA with the slanted microchannel, we achieved fluorescence-activated droplet sorting of 1 nL droplets at a record high throughput of 1752 droplets/s, twice as high as the previously reported maximum sorting throughput of 1 nL droplets.

Canine mammary cancer in overweight or obese female dogs is associated with intratumoral microvessel density and macrophage counts.

Obesity is a major health condition owing to its effects on chronic diseases and cancers in humans, but little information is available regarding the role of obesity in canine mammary cancer (CMC). In the present study, we performed immunohistochemistry to investigate the effect of obesity on CMC by analyzing the number of tumor-associated macrophages, intratumoral microvessel density (iMVD), and the expression of prognostic factors including epidermal growth factor receptor (EGFR), cyclooxygenase 2 (COX-2), and Ki67 in CMC specimens. These data were compared in CMC specimens from lean or ideal body weight (Group 1) versus overweight or obese (Group 2) female dogs (n = 60 for each group). Associations between obesity status and histologic characteristics, such as histologic subtype, grading, and lymphatic invasion, were also investigated. Compared with lean or ideal body weight dogs, TAM (tumor-associated macrophage) counts (P < .005) and iMVD (P < .001) were significantly higher in overweight or obese dogs. CMC specimens of dogs in the overweight or obese group also showed higher histologic grade (P < .001). In addition, although no association was found between obesity status and either COX-2 or EGFR expression, Ki67 expression was greater in CMC specimens of overweight or obese dogs (P < .005). The results of this study suggest that obesity may influence CMC development and progression, being associated with higher histologic grade, greater infiltration of TAMs, and increased tumor angiogenesis.

Surface-Enhanced Raman Scattering-Based Dual-Flow Lateral Flow Assay Sensor for the Ultrasensitive Detection of the Thyroid-Stimulating Hormone.

The surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) strip has been considered a high-sensitivity sensor that can overcome the low sensitivity and the difficulty of quantitative analysis problems inherent in the colorimetric LFA sensor. In the SERS-based LFA strip reported so far, a liquid sample flows through the nitrocellulose membrane in a single pathway. In some cases, however, this single-flow approach still has a limitation in detection sensitivity. This study developed a conceptually new SERS-based dual-flow LFA sensor to improve the detection sensitivity in a single-flow LFA sensor. First, a 25 nm Raman reporter-labeled gold nanoparticle (AuNP) solution flowed through one way, and a 45 nm AuNP solution continuously flowed through another path. This sequential flow of two different AuNP solutions enables forming additional bright hot spots between 25 and 45 nm AuNPs in the test line, and the SERS signal is strongly enhanced. Using this SERS-based dual-flow LFA sensor, it was possible to detect thyroid-stimulating hormone less than 0.5 µIU/mL that cannot be measured with a SERS-based single-flow LFA sensor.

EGFR Overexpression and Sequence Analysis of KRAS, BRAF, and EGFR Mutation Hot Spots in Canine Intestinal Adenocarcinoma.

Epidermal growth factor receptor (EGFR) is overexpressed in many human colorectal cancers and anti-EGFR agents are employed as immunotherapies. However, KRAS, EGFR, and BRAF gene mutations can influence the activity of the anti-EGFR agents. We evaluated EGFR expression at protein and mRNA levels in canine intestinal adenocarcinomas using immunohistochemistry (IHC) and RNA in situ hybridization (RNA-ISH). We also investigated the mutation status of EGFR, KRAS, and BRAF to aid the development of anti-EGFR agents for canine intestinal adenocarcinoma. EGFR expression was highest in adenocarcinoma, followed by intramucosal neoplasia (adenoma and in situ carcinoma), and nonneoplastic canine intestinal tissue, at both protein (P = .000) and mRNA (P = .005) levels. The EGFR, KRAS, and BRAF genes showed wild-type sequences at the mutation hot spots in all 13 specimens. Thus, EGFR might serve as a promising diagnostic marker in canine intestinal adenocarcinoma, and further studies would be needed to develop EGFR-targeted anticancer therapies.

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b'a' Domain.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b'a' domain of protein disulfide isomerase (PDIb'a') greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb'a'-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.

Ferroelectric-mediated filamentary resistive switching in P(VDF-TrFE)/ZnO nanocomposite films.

In ferroelectric (FE) polymer-semiconducting polymer blend based organic resistive random access memory devices (ReRAM), the carriers are injected into the semiconductor region of the blend because of the polarization originated internal electric field in the FE polymer. A higher concentration of semiconducting polymer in the FE polymer-semiconducting polymer blends usually generate a high leakage current and degrades the FE characteristics of the FE polymer resulting in a high OFF current and consequently a low ON/OFF ratio. In order to achieve a high ON/OFF ratio in the FE polymer/semiconducting polymer blends, the FE properties of the FE polymer should be preserved. In this study, organic ReRAMs based on ferroelectric poly(vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)) and ZnO nanoparticle (NPs) blends exhibiting bipolar resistive switching and a high ON/OFF ratio were realized using a low-cost solution process. Unlike conventional ferroelectric polymer-semiconducting polymer blend systems where FE characteristics are suppressed in ReRAMs, our Au/P(VDF-TrFE)_ZnO NPs/n++Si devices retain the FE characteristics of the P(VDF-TrFE) polymers. Our devices switch between bi-stable resistance states via the ferroelectric-assisted filamentary conduction mechanism. Based on ex situ transmission electron microscopy and elemental mapping analyses, we found that the resistive switching occurs through the formation of conduction paths consisting of Zn-rich/F-deficient regions. The device fabricated at a blend ratio of 20 wt% ZnO NPs in P(VDF-TrFE) matrix exhibited optimal stable resistive switching behavior with an ON/OFF ratio of up to 2 × 107 and a retention time of 104 s.

CD204-Expressing Tumor-Associated Macrophages Are Associated With Malignant, High-Grade, and Hormone Receptor-Negative Canine Mammary Gland Tumors.

Tumor-associated macrophages (TAMs) are an important component of leukocyte infiltration in tumors. TAMs can be classified into M1 and M2 phenotypes. In the present study, the expression of CD204, an M2-polarized macrophage receptor, was investigated by immunohistochemistry in the area surrounding TAMs in 101 cases of canine mammary gland tumor (CMT). We examined the relationship between M2-polarized TAMs and malignancy, histological subtype, histological grade, molecular subtype, hormone receptor (HR) status, and clinical obesity indices. The mean number of CD204-positive macrophages was significantly higher in malignant CMTs than in benign CMTs ( P = .000). The number of CD204-positive macrophages differed significantly between histological grades ( P = .000) and were significantly higher in grade III than in grades I and II. Moreover, the mean number of CD204-positive macrophages was significantly higher in HR-negative malignant CMTs than in HR-positive malignant CMTs ( P = .035) and in malignant CMTs with lymphatic invasion compared to malignant CMTs without lymphatic invasion ( P = .000). These findings suggest that CD204-positive macrophages might affect the development and behavior of CMTs and highlight the potential of CD204 as a prognostic factor.

Coronary artery atherosclerosis associated with shift work in chemical plant workers by using coronary CT angiography.

OBJECTIVES: The aim of this study was to investigate whether shift work is related to elevated risk of coronary artery disease (CAD) by determining the coronary artery calcium (CAC) score and the presence of coronary artery stenosis by using coronary artery CT angiography (CCTA). METHODS: In this study, 110 male workers participated and underwent a CCTA examination for CAC scoring, which represents coronary artery plaque, and were evaluated for luminal stenosis. All of the participants were working in the same chemical plant, of whom 70 worked day shifts and 40 worked rotating shifts. RESULTS: In a multivariate logistic regression analysis, including age, smoking status, alcohol consumption, regular exercise and waist circumference, shift work was associated with a 2.89-fold increase in the odds of developing coronary plaque compared with day work (OR, 2.89; 95% CI 1.07 to 7.82). The association between shift work and coronary plaque was strong after adjustment for age, low-density lipoprotein cholesterol, hypertension and diabetes mellitus (OR, 2.92; 95% CI 1.02 to 8.33). In addition, the number of years of shift work employment was associated with coronary plaque. However, no association was found between shift work and coronary artery stenosis. CONCLUSIONS: Shift work could induce CAD onset via the atherosclerotic process, and shift work employment duration was associated with an increased risk of atherosclerosis in male workers.

Antagonism of VEGF-A-induced increase in vascular permeability by an integrin α3ß1-Shp-1-cAMP/PKA pathway.

In cancer, VEGF-induced increase in vascular permeability results in increased interstitial pressure, reducing perfusion and increasing hypoxia, which reduce delivery of chemotherapeutic agents and increase resistance to ionizing radiation. Here, we show that both TIMP-2 and Ala + TIMP-2, a TIMP-2 mutant without matrix metalloproteinase inhibitory activity, antagonize the VEGF-A-induced increase in vascular permeability, both in vitro and in vivo. Like other agents known to preserve endothelial barrier function, TIMP-2 elevates cytosolic levels of cAMP and increases cytoskeletal-associated vascular endothelial cadherin in human microvascular endothelial cells. All of these effects are completely ablated by selective knockdown of integrin α3ß1 expression, expression of a dominant negative protein tyrosine phosphatase Shp-1 mutant, administration of the protein tyrosine phosphatase inhibitor orthovanadate, or the adenylate cyclase inhibitor SQ22536. This TIMP-2-mediated inhibition of vascular permeability involves an integrin α3ß1-Shp-1-cAMP/protein kinase A-dependent vascular endothelial cadherin cytoskeletal association, as evidenced by using siRNAs to integrin α3ß1 and Shp-1, or treatment with Shp-1 inhibitor NSC87877 and protein kinase A inhibitor H89. Our results demonstrate the potential utility for TIMP-2 in cancer therapy through "normalization" of vascular permeability in addition to previously described antiangiogenic effects.

Transcriptomic profiling analysis of the effect of palmitic acid on 3D spheroids of ß-like cells derived from induced pluripotent stem cells.

Type 2 diabetes (T2D) is posing a serious public health concern with a considerable impact on human life and health expenditures worldwide. The disease develops when insulin plasma level is insufficient for coping insulin resistance, caused by the decline of pancreatic ß-cell function and mass. In ß-cells, the lipotoxicity exerted by saturated free fatty acids in particular palmitate (PA), which is chronically elevated in T2D, plays a major role in ß-cell dysfunction and mass. However, there is a lack of human relevant in vitro model to identify the underlying mechanism through which palmitate induces ß-cell failure. In this frame, we have previously developed a cutting-edge 3D spheroid model of ß-like cells derived from human induced pluripotent stem cells. In the present work, we investigated the signaling pathways modified by palmitate in ß-like cells derived spheroids. When compared to the 2D monolayer cultures, the transcriptome analysis (FDR set at  0.1) revealed that the 3D spheroids upregulated the pancreatic markers (such as GCG, IAPP genes), lipids metabolism and transporters (CD36, HMGSC2 genes), glucose transporter (SLC2A6). Then, the 3D spheroids are exposed to PA 0.5 mM for 72 h. The differential analysis demonstrated that 32 transcription factors and 135 target genes were mainly modulated (FDR set at  0.1) including the upregulation of lipid and carbohydrates metabolism (HMGSC2, LDHA, GLUT3), fibrin metabolism (FGG, FGB), apoptosis (CASP7). The pathway analysis using the 135 selected targets extracted the fibrin related biological process and wound healing in 3D PA treated conditions. An overall pathway gene set enrichment analysis, performed on the overall gene set (with pathway significance cutoff at 0.2), highlighted that PA perturbs the citrate cycle, FOXO signaling and Hippo signaling as observed in human islets studies. Additional RT-PCR confirmed induction of inflammatory (IGFBP1, IGFBP3) and cell growth (CCND1, Ki67) pathways by PA. All these changes were associated with unaffected glucose-stimulated insulin secretion (GSIS), suggesting that they precede the defect of insulin secretion and death induced by PA. Overall, we believe that our data demonstrate the potential of our spheroid 3D islet-like cells to investigate the pancreatic-like response to diabetogenic environment.

Dynamic, IPSC-derived hepatic tissue tri-culture system for the evaluation of liver physiologyin vitro.

Availability of hepatic tissue for the investigation of metabolic processes is severely limited. While primary hepatocytes or animal models are widely used in pharmacological applications, a change in methodology towards more sustainable and ethical assays is highly desirable. Stem cell derived hepatic cells are generally regarded as a viable alternative for the above model systems, if current limitations in functionality and maturation can be overcome. By combining microfluidic organ-on-a-chip technology with individually differentiated, multicellular hepatic tissue fractions, we aim to improve overall functionality of hepatocyte-like cells, as well as evaluate cellular composition and interactions with non-parenchymal cell populations towards the formation of mature liver tissue. Utilizing a multi-omic approach, we show the improved maturation profiles of hepatocyte-like cells maintained in a dynamic microenvironment compared to standard tissue culture setups without continuous perfusion. In order to evaluate the resulting tissue, we employ single cell sequencing to distinguish formed subpopulations and spatial localization. While cellular input was strictly defined based on established differentiation protocols of parenchyma, endothelial and stellate cell fractions, resulting hepatic tissue was shown to comprise a complex mixture of epithelial and non-parenchymal fractions with specific local enrichment of phenotypes along the microchannel. Following this approach, we show the importance of passive, paracrine developmental processes in tissue formation. Using such complex tissue models is a crucial first step to develop stem cell-derivedin vitrosystems that can compare functionally with currently used pharmacological and toxicological applications.

Ultra-high density protein spots achieved by on chip digitalized protein synthesis.

Current methodologies for arraying proteins using cell-free protein synthesis on a chip have spatial limitations that prevent reaching ultra-high density necessary for high throughput analysis. To circumvent this, we developed an on-chip method based on microcompartmentalization of protein synthesis. Proteins are synthesized in arrayed micrometer scale chambers from confined DNA template molecules. On-chip protein expression is highly efficient and the method can be used with a minimal amount of template i.e. single DNA molecules to perform digitalized cell-free protein synthesis (d-CFPS). A functionalized surface at the floor of the tightly sealed microchambers enables direct capture of expressed proteins. A density of 104 spots per mm² was achieved, which represents a gain by more than 3 orders of magnitude over conventional methods. This technique of forming such densely arrayed small protein spots is the first step towards the development of a general method that would allow fabrication of ultra-high density protein arrays for high-throughput analysis.

An eco-friendly treatment of tannery wastewater using bioaugmentation with a novel microbial consortium.

A novel microbial consortium (BM-S-1) enriched from natural soils was successfully used to treat tannery wastewater from leather manufacturing industries in Korea on a pilot scale. The objective of this study was to determine whether augmentation with a novel microbial consortium BM-S-1could successfully treat the recalcitrant wastewater without chemical pre-treatment in a tannery wastewater treatment system. Chemical oxygen demand (COD), total nitrogen (TN) and total phosphorus (TP) were monitored for water quality. The microbial population dynamics were analyzed using pyrosequencing, and denitrifying bacteria were quantified using real-time PCR (RT-PCR). The removal efficiencies for COD, TN and TP were greater than 91%, 79%, and 90%, respectively. The dominant phyla in the buffering tank (B), primary aeration (PA), secondary aeration (SA) and sludge digestion tank (SD) were Proteobacteria, Firmicutes, Bacteroidetes, Planctomycetes and Deinococcus-Thermus. Cluster analysis based on the UniFrac distance of the species in the different stages showed that the PA is similar to the SA, whereas the B is similar to the SD. qPCR of the nosZ genes showed the highest abundance of denitrifiers in B, which was increased 734-fold compared to the influent (I). It was hypothesized that anaerobic denitrifiers and the diverse microbial community may play important roles in the biological treatment of tannery wastewater. This technology may also contribute to the full-scale treatment of industrial wastewater containing food processing wastewater and marine sediment with high organic content.

Electrochemical response of surface-attached redox DNA governed by low activation energy electron transfer kinetics.

The mechanism responsible for electron transport within layers of redox DNA anchored to electrodes has been extensively studied over the last twenty years, but remains controversial. Herein, we thoroughly study the electrochemical behavior of a series of short, model, ferrocene (Fc) end-labeled dT oligonucleotides, terminally attached to gold electrodes, using high scan rate cyclic voltammetry complemented by molecular dynamics simulations. We evidence that the electrochemical response of both single-stranded and duplexed oligonucleotides is controlled by the electron transfer kinetics at the electrode, obeying Marcus theory, but with reorganization energies considerably lowered by the attachment of the ferrocene to the electrode via the DNA chain. This so far unreported effect, that we attribute to a slower relaxation of water around Fc, uniquely shapes the electrochemical response of Fc-DNA strands and, being markedly dissimilar for single-stranded and duplexed DNA, contributes to the signaling mechanism of E-DNA sensors.

Single-Cell Electrochemical Aptasensor Array.

Despite several demonstrations of electrochemical devices with limits of detection (LOD) of 1 cell/mL, the implementation of single-cell bioelectrochemical sensor arrays has remained elusive due to the challenges of scaling up. In this study, we show that the recently introduced nanopillar array technology combined with redox-labeled aptamers targeting epithelial cell adhesion molecule (EpCAM) is perfectly suited for such implementation. Combining nanopillar arrays with microwells determined for single cell trapping directly on the sensor surface, single target cells are successfully detected and analyzed. This first implementation of a single-cell electrochemical aptasensor array, based on Brownian-fluctuating redox species, opens new opportunities for large-scale implementation and statistical analysis of early cancer diagnosis and cancer therapy in clinical settings.