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The downregulation of melatonin receptor 1A (MTNR1A) is associated with a range of pathological conditions, including membranous nephropathy. Knowledge of the mechanism underlying MTNR1A expression has been limited to the transcriptional regulation level. Here, RNA interference screening in human kidney cells revealed that heterogeneous nuclear ribonucleoprotein L (hnRNPL) upregulated MTNR1A RNA post-transcriptionally. hnRNPL knockdown or overexpression led to increased or decreased levels of cyclic adenosine monophosphate-responsive element-binding protein phosphorylation, respectively. Molecular studies showed that cytoplasmic hnRNPL exerts a stabilizing effect on the MTNR1A transcript through CA-repeat elements in its coding region. Further studies revealed that the interaction between hnRNPL and MTNR1A serves to protect MNTR1A RNA degradation by the exosome component 10 protein. MTNR1A, but not hnRNPL, displays a diurnal rhythm in mouse kidneys. Enhanced levels of MTNR1A recorded at midnight correlated with robust binding activity between cytoplasmic hnRNPL and the MTNR1A transcript. Both hnRNPL and MTNR1A were decreased in the cytoplasm of tubular epithelial cells from experimental membranous nephropathy kidneys, supporting their clinical relevance. Collectively, our data identified cytoplasmic hnRNPL as a novel player in the upregulation of MTNR1A expression in renal tubular epithelial cells, and as a potential therapeutic target.

Melatonin is a hormone majorly secreted by the pineal gland and contributes to a various type of physiological functions in mammals. The melatonin production is tightly limited to the AANAT level, yet the most known molecular mechanisms underlying AANAT gene transcription is limited in the pinealocyte. Here, we find that c-Fos and cAMP-response element-binding protein (CREB) decreases and increases the AANAT transcriptional activity in renal tubular epithelial cell, respectively. Notably, c-Fos knockdown significantly upregulates melatonin levels in renal tubular cells. Functional results indicate that AANAT expression is decreased by c-Fos and resulted in enhancement of cell damage in albumin-injury cell model. We further find an inverse correlation between c-Fos and AANAT levels in renal tubular cells from experimental membranous nephropathy (MN) samples and clinical MN specimens. Our finding provides the molecular basis of c-Fos in transcriptionally downregulating expression of AANAT and melatonin, and elucidate the protective role of AANAT in preventing renal tubular cells death in albumin-injury cell model and MN progression.

Plants have evolved diverse cellular mechanisms to maintain their fitness under stressful environments. Banana (Musa spp.) plants inoculated with a new rhizobacterial isolate, Pseudomonas aeruginosa strain Y1 (PaY1), showed improved vegetative growth, and increased levels of H2O2, glucosinolates, phenolic compounds, and lignin contents. Plants treated with PaY1 had reduced membrane peroxidation and electrolyte leakages, and exhibited higher PSII efficiencies after drought and submergence treatments. Furthermore, PaY1 treatment reduced disease infection rates in the Foc TR4 (Fusarium wilt pathogen)-inoculated plants. RNA seq results identified signaling molecules activated by PaY1, including ROS/antioxidant molecules and signaling of G protein and sphingolipids. Besides, PaY1 exerted positive effects on hormonal signaling, including auxin, ABA, and jasmonic acid (JA) in banana seedlings. Genes associated with cellular pathways of cell division and flowering control were significantly affected; up-regulated genes were also implicated in cellular pathways regulating nutrient availability. The up-regulated genes functioning in abiotic stress tolerance comprised genes encoding DETOXIFICATION-like proteins, aquaporins, autophagy-like, and MAPKK2-like. Furthermore, genes encoding proteins participating in the regulatory network of disease resistance were discovered, which included members of pathogenesis-related proteins (PRs), cell death associated proteins, proteins implicated in the MAMP-trigger immunity (MTI), and genes such as RGA2-like and ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR 1B (ERF1B) with predicted functions regulating disease resistance against Fusarium oxysporum in different plant species. The transcriptome data presented here indicated that PaY1 is a potent activator of banana genes related to hormonal signaling and cellular pathways including antioxidant defense, detoxification, and MTI, which worked collaboratively to control plant growth, abiotic and biotic stress tolerance in banana plants.

The effective and efficient treatment of diseases, such as HIV, cancer or hereditary diseases, requires accurate and precise control of the combinatorial drug-dosage and their release. Herein, we introduce a simple photosensitive poly(ethylene glycol) diacrylate (PEGDA) hydrogel based platform for high dynamic range testing of combinatorial cocktail drug screening using three chemical and two protein drug treatments for colon cancer. UV cross linked PEGDA hydrogel droplet arrays on a Teflon patterned glass substrate enable a rapid yet accurate selection and dosage assignment of the drugs. Precisely loaded cocktails of the anticancer drugs were simultaneously released in-parallel with the PEGDA hydrogel chips into 2D or 3D cultured HCT-8 colon cancer cells for combinatorial drug screening. We demonstrate the functionality of our UV gel-Drug Chips 1000 fold range of concentrations for each of the five drugs in 30 seconds to find the optimized drug cocktail using a fractional factorial control system. Our device has low drug consumption, requiring only 12 nL per screening run per droplet. In addition, our UV gel-Drug Chips were employed for find the optimized drug cocktail using a fractional search algorithm. Our cocktail drug response results for both 2D (cell viability is 7.3%) and 3D (cell viability is 10.8%) colon cancer cells were analogous to those found by conventional method (6.8 and 9.3 respectively). In contrast to conventional method, our approach is faster, more effective, less time consuming and requires a lower amounts of drug volume.

Idiopathic membranous nephropathy (MN) is an autoimmune-mediated glomerulonephritis and the most common cause of idiopathic nephrotic syndrome in adult humans. A tumor necrosis factor α (TNF-α)-mediated inflammatory response via TNF receptor 1 (TNFR1) and TNFR2 has been proposed as a pathogenic factor. In this study, we assessed the therapeutic response to blocking TNF signaling in experimental MN. Murine MN was induced experimentally by cationic bovine serum albumin (cBSA); phosphate-buffered saline was used in control mice. In MN mice, TNF was inhibited by etanercept blocking of TNFR1/TNFR2 or the preligand assembly domain fusion protein (PLAD.Fc), a small fusion protein that can preferentially block TNFR1 signaling. Disease severity and possible mechanisms were assessed by analyzing the metabolic and histopathology profiles, lymphocyte subsets, immunoglobulin production, oxidative stress, and apoptosis. cBSA-induced MN mice exhibited typical nephrotic syndrome and renal histopathology. MN mice given etanercept or PLAD.Fc did not exhibit significant reduction of proteinuria, amelioration of glomerular lesions, or attenuation of immune complex deposition. Immune cell subsets, serum immunoglobulin levels, production of reactive oxygen species, and cell apoptosis in the kidney were not altered by TNF inhibition. By contrast, MN mice receiving etanercept or PLAD.Fc exhibited significantly decreased infiltration of immune cells into the kidney. These results show that the therapeutic effects of blocking TNFR1 and/or TNFR2 signaling in experimental MN are not clinically effective. However, TNF signaling inhibition significantly attenuated renal immune cell infiltration in experimental MN.

The potential probiotic properties of lactic acid bacteria (LAB) after treatment with gastrointestinal (GI) conditions were investigated. Some LAB strains that survived simulated GI treatment retained their adhesiveness and antagonism against the pathogen. Therefore pre-challenging LAB with simulated GI conditions is a suitable way for potential probiotic studies.

Cell alignment is a critical factor to govern cellular behavior and function for various tissue engineering applications ranging from cardiac to neural regeneration. In addition to physical geometry, strain is a crucial parameter to manipulate cellular alignment for functional tissue formation. In this paper, we introduce a simple approach to generate a range of gradient static strains without external mechanical control for the stimulation of cellular behavior within 3D biomimetic hydrogel microenvironments. A glass-supported microfluidic chip with a convex flexible polydimethylsiloxane (PDMS) membrane on the top was employed for loading the cells suspended in a prepolymer solution. Following UV crosslinking through a photomask with a concentric circular pattern, the cell-laden hydrogels were formed in a height gradient from the center (maximum) to the boundary (minimum). When the convex PDMS membrane retracted back to a flat surface, it applied compressive gradient forces on the cell-laden hydrogels. The concentric circular hydrogel patterns confined the direction of hydrogel elongation, and the compressive strain on the hydrogel therefore resulted in elongation stretch in the radial direction to guide cell alignment. NIH3T3 cells were cultured in the chip for 3 days with compressive strains that varied from ~65% (center) to ~15% (boundary) on hydrogels. We found that the hydrogel geometry dominated the cell alignment near the outside boundary, where cells aligned along the circular direction, and the compressive strain dominated the cell alignment near the center, where cells aligned radially. This study developed a new and simple approach to facilitate cellular alignment based on hydrogel geometry and strain stimulation for tissue engineering applications. This platform offers unique advantages and is significantly different than the existing approaches owing to the fact that gradient generation was accomplished in a miniature device without using an external mechanical source.

The integrated effect of the cholesterol and CYP46 genotypes on the risk of cognitive decline needs to be determined. Using the Cognitive Abilities Screening Instrument (CASI), 145 mentally healthy middle-aged and older adults were recruited to investigate the influence of cholesterol and CYP46 genotypes on cognitive event-related potentials (ERPs). The subjects with a high low-density lipoprotein cholesterol (LDL-C) level displayed significantly lower amplitude ERPs, although the CASI scores showed no difference. There was no association between the CYP46 genotypes, CASI scores, cholesterol levels, and measures of ERPs. No interaction between LDL-C level and CYP46 genotypes was noted. The LDL-C level is an independent predictor of low P300 amplitude. Prevention and treatment of high cholesterol may be of potential benefit in reducing cognitive impairment.

Membranous nephropathy (MN), a type of glomerular nephritis, is one of the most common causes of nephrotic syndrome in adults. Although it is known that melatonin plays a protective role in MN, the role of melatonin receptors in the pathophysiology of MN is unclear. Using an experimental MN model and clinical MN specimens, we studied melatonin receptor expression and found that melatonin receptor 1A (MTNR1A) expression was significantly downregulated in renal tubular epithelial cells. Molecular studies showed that the transcription factor pituitary homeobox-1 (PITX1) promoted MTNR1A expression via direct binding to its promoter. Treatment of a human tubular cell line with albumin to induce injury resulted in the stable reduction in MTNR1A and PITX1 expression. PITX1 levels were significantly downregulated in tubular epithelial cells from mice MN kidneys and MN renal specimens. Knockdown of MTNR1A, PITX1, or cyclic adenosine monophosphate-responsive element-binding protein (CREB) decreased E-cadherin (CDH1) expression, but upregulated Per2 and α-smooth muscle actin (αSMA) expression. Blockade of the MTNR1A receptor with luzindole in MN mice further impaired renal function; this was accompanied by CDH1 downregulation and Per2 and αSMA upregulation. Together, our results suggest that in injured tissue, decreased PITX1 expression at the MTNR1A promoter regions leads to decreased levels of MTNR1A in renal tubular epithelial cells, which increases the future risk of MN.

Protein microarrays are emerging as an important enabling technology for the simultaneous investigation of complicated interactions among thousands of proteins. The solution-based blocking protocols commonly used in protein microarray assays often cause cross-contamination among probes and diminution of protein binding efficiency because of the spreading of blocking solution and the obstruction formed by the blocking molecules. In this paper, an alternative blocking process for protein microarray assays is proposed to obtain better performance by employing a vapor-phase deposition method to form self-assembled surface coatings using a highly fluorinated organosilane as the blocking agent on the background surfaces. Compared to conventional solution-based blocking processes, our experimental results showed that this vapor-phase process could shorten the blocking time from hours to less than 10 min, enhance the binding efficiency by up to 6 times, reduce the background noise by up to 16 times, and improve the S/N ratio by up to 64 times. This facile blocking process is compatible with current microarray assays using silica-based substrates and can be performed on many types of silane-modified surfaces.

We reports an approach to fabricate dual-faced polystyrene beads (DFPSBs) with tri-functions of tumor cell recognition, drug delivery, and real-time Raman sensing. Onestep oxygen-plasma treatment process was used to etch commercially available fluorescent polystyrene beads into a corrugated upper hemisphere and simultaneously change the entire surface with carboxylic groups. After depositing gold onto the corrugated hemisphere for surface enhanced Raman scattering (SERS) while leaving the other smooth and clean hemisphere for fluorescence detection, the DFPSBs are formed with dual-surfaces of plasmonic gold semishells on the top and fluorescent carboxylated polystyrene at the bottom. Sulfo-NHS-SS-biotin disulfide linkers and anti-CD44 antibodies can be modified and added onto the top gold surfaces and the bottom carboxyl groups through Au-S and peptide bonds, respectively. Then, the surface-modified DFPSB suspension can be employed to target overexpressive glycoproteins (CD44) on the surfaces of cancer cells and release their loads via the cleavage of disulfide bonds in the cytoplasm environment. These anti-CD44-modified DFPSBs exhibit a 12-fold cancer targeting ability on HeLa cells when compared to a normal chondrocyte cell.

The Surface Enhanced Raman Scattering (SERS) bacteria sensing chip was usually developed by wet-etching [1] or template-molding [2] approach to obtain an enhancement factor of 101−3. However, these processes may not be available for the template and difficult to integrate with other complicated fabrication designs. Therefore, in this project, we propose an easy integrated approach to develop high sensitive surface enhanced Raman scattering (SERS) with an enhancement factor of 105−6 via a metal-coated polystyrene array for quick detection of the specific bacteria in food. In the experimental results, it is detectable of less than five bacteria per laser spot (∼3 μm in diameter) on the silver-coated corrugated polystyrene bead array. The goal of this project is also to develop a disposable and room temperature fabricated SERS sensors for future integration.

Many of SERS nanoparticles took advantage of the surface roughness for the significant improvement of their Raman sensing ability. Nevertheless, few papers analyzed the characteristics of surface roughness nanostructures that contribute to the SERS. Thus, this paper investigates the characteristics of the corrugated polystyrene bead (PSB) array etched by a series of oxygen plasma etching time for giving a criterion to fabricate appropriate SERS-active nanoparticles. Three factors were considered in this paper: (1) the effect of plasma coupling among neighboring particles, (2) the vertical surface roughness of nanocorrugations, and (3) the pitch size, the lateral surface roughness, of nanocorrugations. By the analysis of SEM and AFM images, those factors were quantifiable. The correlation coefficient between each factor and SERS Raman enhancement was also investigated to verify that the pitch size of nanocorrugations (ranging from ∼6 nm to ∼12 nm on the surface of PSBs) dominates the SERS enhancement. Therefore, the maximum improvement of Raman intensity that derives from surface roughness treatment is 12 times compared to smooth surface. Moreover, it has a high enhancement factor of ∼106.

This paper describes a Self-Aligned Wet (SAW) cell suitable for direct-cell or bacteria incubation and observation in a wet environment inside a transmission electron microscope. This SAW cell is fabricated by a bulk-micromachining process and composed of two structurally complementary counterparts (an out-frame and an in-frame), where each contain a silicon nitride film based observation window. The in- and out-frames can be self-aligned via a mechanism of surface tension from a bio-sample droplet without the aid of positioning stages. The liquid chamber is enclosed between two silicon nitride membranes that are thin enough to allow high energy electrons to penetrate while also sustaining the pressure difference between the TEM vacuum and the vapor pressure within the liquid chamber. A large field of view (150 μm × 150 μm) in a SAW cell is favored and formed from a larger sized observation window in the out-frame, which is fabricated using a unique circular membrane formation process. In this paper, we introduce a novel design to circumvent the challenges of charging/heating problems in silicon nitride that arise from interactions with an electron beam. This paper also demonstrates TEM observations of D. Radiodurans growth in a liquid environment within a thicker chamber (20 μm) within a SAW cell.

This paper reports the employment of carboxyl groups on polystyrene beads surface to generate high aspect ratio (HSR) nano-extrutions by plasma etching for enhancing SERS signal. We investigate the detail mechanism of the surface corrugation formation on PSBs and propose a novel method to produce HSR surface nano-extrutions on carboxyl-rich PSBs surface. By understanding the mechanism of the surface corrugation formation, we can control the HSR. Therefore, a 4-fold Raman signal enhancement was achieved when compared to the result by previous method, using oxygen plasma to generate corrugation surface on PSBs without carboxyl groups.

Although the stability of the nanobubble remains a controversial issue that is subject to the classical predictions of high Laplace pressure, we demonstrate that a hydrogen nanobubble can be generated and stabilized in an aqueous solution of Keyhole limpet hemocyanin (KLH) protein via an electron radiolysis process. The hydrogen gas inside the nanobubble is in a “dense gas” phase that is characterized by a Knudsen number and number density of hydrogen molecules. The dynamics of nanobubbles are analyzed using time-series electron microscopy images. The growth of small nanobubbles will be affected by the largest neighboring nanobubble; however, a diffusive shielding effect for small nanobubbles is observed. Locally, anti-Ostwald ripening of nanobubbles can be observed; however, the global growth behavior among the nanobubbles is randomly correlated because the characteristic diffusion length of the hydrogen molecules is considerably greater than the average spacing among the nanobubbles.

Smart nanocarriers with high SERS enhancement are inevitably important in cancer therapy for treating cells and recording their changes at the single-molecule scale. Therefore, this work reports an approach to fabricating and modifying mass-produced dual-faced and tri-functional nanoparticles for sequentially recognizing tumor cells, drug delivery, and real-time monitoring of biological responses. A one-step oxygen plasma process was employed to tailor commercially available fluorescent polystyrene beads into a corrugated upper hemisphere and simultaneously modify the entire surface with carboxylic groups. After depositing gold onto the corrugated hemisphere for SERS (using 633 nm laser excitation) while leaving the other smooth and clean hemisphere for fluorescence detection (via 488 nm or 532 nm laser excitation), the Au-coated fluorescent nano-mushrooms (AuFNMs) are formed with dual-surfaces of plasmonic gold semishells on the top and fluorescent carboxylated polystyrene at the bottom. Based on the unique structures and the self-generated functional surfaces of the AuFNMs, sulfo-NHS–SS–biotin disulfide linkers and anti-CD44 monoclonal antibodies can be simultaneously modified and added onto the top gold surfaces and the bottom carboxyl groups through Au–S and peptide bonds, respectively. The surface-modified AuFNM suspension, with >99% purity and uniform particle size in a concentration of ∼1010 particles per mL in 2 mL DI water, can be employed to target overexpressive glycoproteins (CD44) on the surfaces of cancer cells and release their loads via the cleavage of disulfide bonds in the cytoplasm. These AuFNMs exhibit a 12-fold higher cancer targeting ability on HeLa cells when compared to a normal chondrocyte cell. Three-dimensional confocal particle tracking and Raman mapping are used to demonstrate the AuFNMs' long-lasting single-particle fluorescence and superior biomolecule sensing ability.