BackgroundAdapter trimming is a prerequisite step for analyzing next-generation sequencing (NGS) data when the reads are longer than the target DNA/RNA fragments. Although typically used in small RNA sequencing, adapter trimming is also used widely in other applications, such as genome DNA sequencing and transcriptome RNA/cDNA sequencing, where fragments shorter than a read are sometimes obtained because of the limitations of NGS protocols. For the newly emerged Nextera long mate-pair (LMP) protocol, junction adapters are located in the middle of all properly constructed fragments; hence, adapter trimming is essential to gain the correct paired reads. However, our investigations have shown that few adapter trimming tools meet both efficiency and accuracy requirements simultaneously. The performances of these tools can be even worse for paired-end and/or mate-pair sequencing.ResultsTo improve the efficiency of adapter trimming, we devised a novel algorithm, the bit-masked k-difference matching algorithm, which has O(kn) expected time with O(m) space, where k is the maximum number of differences allowed, n is the read length, and m is the adapter length. This algorithm makes it possible to fully enumerate all candidates that meet a specified threshold, e.g. error ratio, within a short period of time. To improve the accuracy of this algorithm, we designed a simple and easy-to-explain statistical scoring scheme to evaluate candidates in the pattern matching step. We also devised scoring schemes to fully exploit the paired-end/mate-pair information when it is applicable. All these features have been implemented in an industry-standard tool named Skewer (https://sourceforge.net/projects/skewer). Experiments on simulated data, real data of small RNA sequencing, paired-end RNA sequencing, and Nextera LMP sequencing showed that Skewer outperforms all other similar tools that have the same utility. Further, Skewer is considerably faster than other tools that have comparative accuracies; namely, one times faster for single-end sequencing, more than 12 times faster for paired-end sequencing, and 49% faster for LMP sequencing.ConclusionsSkewer achieved as yet unmatched accuracies for adapter trimming with low time bound.

When nanoparticles are exposed to a physiological environment, a "protein corona" is formed that greatly determines their biological fate. Adsorption of proteins could be influenced by chiral surfaces of nanoparticles; however, very few quantitative studies are available on the interaction of protein with the chiral surface of nanoparticles, and the underlying mechanism remains largely unresolved. We have developed a strategy to quantitatively analyze the adsorption and conformational features of transferrin on gold nanoparticles that are functionalized with d, l, and racemic penicillamine. We used a quartz microbalance platform to monitor the interaction of the adsorbed transferrin with transferrin receptors in HEK cell-derived liposomes. Results show that the chiral surface of nanoparticle determines the orientation and conformation of transferrin, which subsequently affects the interaction and recognition of transferrin with its receptor on the cellular membrane. Transferrin is widely used as a tumor-targeting ligand in cancer treatment and diagnosis since the transferrin receptor is overexpressed on the cell membrane of various types of cancer cells. Thus, the present results will help to expand the knowledge on biological identity of nanoparticles with chiral surfaces in a physiological environment and provide an insight into the rational design of therapeutic nanoparticles.

Chiral properties of nanoscale materials are of importance as they dominate interactions with proteins in physiological environments; however, they have rarely been investigated. In this study, a systematic investigation is conducted for the adsorption behaviors of bovine serum albumin (BSA) onto the chiral surfaces of gold nanoparticles (AuNPs), involving multiple techniques and molecular dynamic (MD) simulation. The adsorption of BSA onto both L- and D-chiral surfaces of AuNPs shows discernible differences involving thermodynamics, adsorption orientation, exposed charges, and affinity. As a powerful supplement, MD simulation provides a molecular-level understanding of protein adsorption onto nanochiral surfaces. Salt bridge interaction is proposed as a major driving force at protein-nanochiral interface interaction. The spatial distribution features of functional groups (COO , NH , and CH ) of chiral molecules on the nanosurface play a key role in the formation and location of salt bridges, which determine the BSA adsorption orientation and binding strength to chiral surfaces. Sequentially, BSA corona coated on nanochiral surfaces affects their uptake by cells. The results enhance the understanding of protein corona, which are important for biological effects of nanochirality in living organisms.

Background/Aims: Contrast induced-acute kidney injury (CI-AKI) is one of the most common causes of acute kidney injury (AKI) in hospitalized patients. Mitophagy, the selective elimination of mitochondria via autophagy, is an important mechanism of mitochondrial quality control in physiological and pathological conditions. In this study, we aimed to determine effects of iohexol and iodixanol on mitochondrial reactive oxygen species (ROS), mitophagy and the potential role of mitophagy in CI-AKI cell models. Methods: Cell viability was measured by cell counting kit-8. Cell apoptosis, mitochondrial ROS and mitochondrial membrane potential were detected by western blot, MitoSOX fluorescence and TMRE staining respectively. Mitophagy was detected by the colocalization of LC3-FITC with MitoTracker Red, western blot and electronic microscope. Results: The results showed that mitophagy was induced in human renal tubular cells (HK-2 cells) under different concentrations of iodinated contrast media. Mitochondrial ROS displayed increased expression after the treatment. Rapamycin (Rap) enhanced mitophagy and alleviated contrast media induced HK-2 cells injury. In contrast, autophagy inhibitor 3-methyladenine (3-MA) down-regulated mitophagy and aggravated cells injury. Conclusions: Together, our finding indicates that iohexol and iodixanol contribute to the generation of mitochondrial ROS and mitophagy. The enhancement of mitophagy can effectively protect the kidney from iodinated contrast (iohexol)-induced renal tubular epithelial cells injury.

As an emerging nanomaterial, graphene quantum dots (GQDs) have shown enormous potential in theranostic applications. However, many aspects of the biological properties of GQDs require further clarification. In the present work, we prepared two sizes of GQDs and for the first time investigated their membrane permeabilities, one of the key factors of all biomedical applications, and transport mechanisms on a Madin Darby Canine Kidney (MDCK) cell monolayer. The experimental results revealed that under ∼300 mg L(-1), GQDs were innoxious to MDCK and did not affect the morphology and integrity of the cell monolayer. The Papp values were determined to be 1-3 × 10(-6) cm s(-1) for the 12 nm GQDs and 0.5-1.5 × 10(-5) cm s(-1) for the 3 nm GQDs, indicating that the 3 nm GQDs are well-transported species while the 12 nm GQDs have a moderate membrane permeability. The transport and uptake of GQDs by MDCK cells were both time and concentration-dependent. Moreover, the incubation of cells with GQDs enhanced the formation of lipid rafts, while inhibition of lipid rafts with methyl-β-cyclodextrin almost eliminated the membrane transport of GQDs. Overall, the experimental results suggested that GQDs cross the MDCK cell monolayer mainly through a lipid raft-mediated transcytosis. The present work has indicated that GQDs are a novel, low-toxic, highly-efficient general carrier for drugs and/or diagnostic agents in biomedical applications.

Cucumber mosaic virus (CMV) infection could induce mosaic symptoms on a wide-range of host plants. However, there is still limited information regarding the molecular mechanism underlying the development of the symptoms. In this study, the coat protein (CP) was confirmed as the symptom determinant by exchanging the CP between a chlorosis inducing CMV-M strain and a green-mosaic inducing CMV-Q strain. A yeast two-hybrid analysis and bimolecular fluorescence complementation revealed that the chloroplast ferredoxin I (Fd I) protein interacted with the CP of CMV-M both in vitro and in vivo, but not with the CP of CMV-Q. The severity of chlorosis was directly related to the expression of Fd1, that was down-regulated in CMV-M but not in CMV-Q. Moreover, the silencing of Fd I induced chlorosis symptoms that were similar to those elicited by CMV-M. Subsequent analyses indicated that the CP of CMV-M interacted with the precursor of Fd I in the cytoplasm and disrupted the transport of Fd I into chloroplasts, leading to the suppression of Fd I functions during a viral infection. Collectively, our findings accentuate that the interaction between the CP of CMV and Fd I is the primary determinant for the induction of chlorosis in tobacco.

The purpose of this study was to develop a scanning electrochemical microscopy (SECM) and scanning electrogenerated chemiluminescence (SECL) setup to visualize the localized enzymatic activity using glucose oxidase as a model. Combination of SECM and electrogenerated chemiluminescence (ECL) was made possible by integrating a photomultiplier tube (PMT) within a SECM setup which is mounted on top of an inverted microscope. An enzyme-polymer spot formed on a glass slide and placed on top of the entrance window of the PMT was used as a model sample to evaluate the potential of the combined SECM/ECL setup. Hydrogen peroxide, which was locally generated by the glucose oxidase (GOx)-catalyzed reaction, reacted with oxidized luminol which was simultaneously electrochemically generated at the positioned SECM electrode tip. By using the phase-sensitive lock-in amplifier, the potential applied to the SECM tip was sinusoidally swept to invoke an associated oscillation of the ECL. Thus, sensitivity of SECL could be substantially enhanced. Images of the local immobilized enzyme activity obtained both by ECL and generator/collector (GC) mode of SECM were compared to elucidate the pathway in which the SECM and SECL signals are generated.

The production of reactive oxygen species (ROS) forms part of the defense reaction of plants against invading pathogens. ROS have multifaceted signaling functions in mediating the establishment of multiple responses. To verify whether hydrogen peroxide (H2 O2 ) contributes to plant virus infection and the development of induced symptoms, we used fluorescence to monitor the generation of H2 O2 and confocal laser scanning microscopy (CLSM) to investigate the subcellular distribution of H2 O2 in leaves. In this study, the M strain of Cucumber mosaic virus (M-CMV) induced heavy chlorotic symptoms in Nicotiana tabacum cv. white burley during systemic infection. Compared with mock-inoculated leaves, H2 O2 accumulation in inoculated leaves increased after inoculation, then decreased after 4 days. For systemically infected leaves that showed chlorotic symptoms, H2 O2 accumulation was always higher than in healthy leaves. Subcellular H2 O2 localization observed using CLSM showed that H2 O2 in inoculated leaves was generated mainly in the chloroplasts and cell wall, whereas in systemically infected leaves H2 O2 was generated mainly in the cytosol. The levels of coat protein in inoculated and systemically infected leaves might be associated with changes in the level of H2 O2 and symptom development. Further research is needed to elucidate the generation mechanism and the relationship between coat protein and oxidative stress during infection and symptom development. Copyright © 2016 John Wiley & Sons, Ltd.