Mechanomemory in protein diffusivity of chromatin and nucleoplasm after force cessation.

It is known that external mechanical forces can regulate structures and functions of living cells and tissues in physiology and diseases. However, after cessation of the force, how structures are altered in response to the dynamics of the chromatin and molecules in the nucleoplasm remains elusive. Here, using single-molecule imaging approaches, we show that exogenous local forces via integrins applied for 2 to 10 min decondensed the chromatin and increased chromatin and nucleoplasm protein mobility inside the nucleus, leading to elevated diffusivity of single protein molecules in the nucleoplasm, tens of minutes after the cessation of force. Diffusion experiments with fluorescence correlation spectroscopy in live single cells show that the mechanomemory in chromatin and nucleoplasm protein diffusivity was regulated by nuclear pore complexes. Protein molecular dynamics simulation recapitulated the experimental findings in live cells and showed that nucleoplasm protein diffusivity was regulated by the number of nuclear pore complexes. The mechanomemory in elevated protein diffusivity of the nucleoplasm after force cessation represents a physical process that reverses protein-protein condensation in phase separation via unjamming of the chromatin. Our findings of mechanomemory in chromatin and nucleoplasm protein diffusivity suggest that the effect of force on the nucleus remains tens of minutes after force cessation and thus is more far-reaching than previously anticipated.

Label-Free Monitoring of Coculture System Dynamics: Probing Probiotic and Cancer Cell Interactions via Infrared Spectroscopic Imaging.

Evaluating the dynamic interaction of microorganisms and mammalian cells is challenging due to the lack of suitable platforms for examining interspecies interactions in biologically relevant coculture conditions. In this work, we demonstrate the interaction between probiotic bacteria (Lactococcus lactis and Escherichia coli) and A498 human cancer cells in vitro, utilizing a hydrogel-based platform in a label-free manner by infrared spectroscopy. The L. lactis strain recapitulated in the compartment system secretes polypeptide molecules such as nisin, which has been reported to trigger cell apoptosis. We propose a mid-infrared (IR) spectroscopic imaging approach to monitor the variation of biological components utilizing kidney cells (A498) as a model system cocultured with bacteria. We characterized the biochemical composition (i.e., nucleic acids, protein secondary structures, and lipid conformations) label-free using an unbiased measurement. Several IR spectral features, including unsaturated fatty acids, ß-turns in protein, and nucleic acids, were utilized to predict cellular response. These features were then applied to establish a quantitative relationship through a multivariate regression model to predict cellular dynamics in the coculture system to assess the effect of nisin on A498 kidney cancer cells cocultured with bacteria. Overall, our study sheds light on the potential of using IR spectroscopic imaging as a label-free tool to monitor complex microbe-host cell interactions in biological systems. This integration will enable mechanistic studies of interspecies interactions with insights into their underlying physiological processes.

Oxygenated Wound Dressings for Hypoxia Mitigation and Enhanced Wound Healing.

Oxygen is a critical factor that can regulate the wound healing processes such as skin cell proliferation, granulation, re-epithelialization, angiogenesis, and tissue regeneration. However, hypoxia, a common occurrence in the wound bed, can impede normal healing processes. To enhance wound healing, oxygenation strategies that could effectively increase wound oxygen levels are effective. The present review summarizes wound healing stages and the role of hypoxia in wound healing and overviews current strategies to incorporate various oxygen delivery or generating materials for wound dressing, including catalase, nanoenzyme, hemoglobin, calcium peroxide, or perfluorocarbon-based materials, in addition to photosynthetic bacteria and hyperbaric oxygen therapy. Mechanism of action, oxygenation efficacy, and potential benefits and drawbacks of these dressings are also discussed. We conclude by highlighting the importance of design optimization in wound dressings to address the clinical needs to improve clinical outcomes.

CAF-1 and Rtt101p function within the replication-coupled chromatin assembly network to promote H4 K16ac, preventing ectopic silencing.

Replication-coupled chromatin assembly is achieved by a network of alternate pathways containing different chromatin assembly factors and histone-modifying enzymes that coordinate deposition of nucleosomes at the replication fork. Here we describe the organization of a CAF-1-dependent pathway in Saccharomyces cerevisiae that regulates acetylation of histone H4 K16. We demonstrate factors that function in this CAF-1-dependent pathway are important for preventing establishment of silenced states at inappropriate genomic sites using a crippled HMR locus as a model, while factors specific to other assembly pathways do not. This CAF-1-dependent pathway required the cullin Rtt101p, but was functionally distinct from an alternate pathway involving Rtt101p-dependent ubiquitination of histone H3 and the chromatin assembly factor Rtt106p. A major implication from this work is that cells have the inherent ability to create different chromatin modification patterns during DNA replication via differential processing and deposition of histones by distinct chromatin assembly pathways within the network.

Monomeric cohesin state revealed by live-cell single-molecule spectroscopy.

The cohesin complex plays an important role in the maintenance of genome stability. Cohesin is composed of four core subunits and a set of regulatory subunits that interact with the core subunits. Less is known about cohesin dynamics in live cells and on the contribution of individual subunits to the overall complex. Understanding the tethering mechanism of cohesin is still a challenge, especially because the proposed mechanisms are still not conclusive. Models proposed to describe tethering depend on either the monomeric cohesin ring or a cohesin dimer. Here, we investigate the role of cohesin dynamics and stoichiometry in live yeast cells at single-molecule resolution. We explore the effect of regulatory subunit deletion on cohesin mobility and found that depletion of different regulatory subunits has opposing effects. Finally, we show that cohesin exists mostly as a canonical monomer throughout the cell cycle, and its monomeric form is independent of its regulatory factors. Our results demonstrate that single-molecule tools have the potential to provide new insights into the cohesin mechanism of action in live cells.

Chromosome loading of cohesin depends on conserved residues in Scc3.

Cohesin is essential for sister chromatid cohesion, which ensures equal segregation of the chromatids to daughter cells. However, the molecular mechanism by which cohesin mediates this function is elusive. Scc3, one of the four core subunits of cohesin, is vital to cohesin activity. However, the mechanism by which Scc3 contributes to the activity and identity of its functional domains is not fully understood. Here, we describe an in-frame five-amino acid insertion mutation after glutamic acid 704 (scc3-E704ins) in yeast Scc3, located in the middle of the second armadillo repeat. Mutated cohesin-scc3-E704ins complexes are unable to establish cohesion. Detailed molecular and genetic analyses revealed that the mutated cohesin has reduced affinity to the Scc2 loader. This inhibits its enrichment at centromeres and chromosomal arms. Mutant complexes show a slow diffusion rate in live cells suggesting that they induce a major conformational change in the complex. The analysis of systematic mutations in the insertion region of Scc3 revealed two conserved aspartic acid residues that are essential for the activity. The study offers a better understanding of the contribution of Scc3 to cohesin activity and the mechanism by which cohesin tethers the sister chromatids during the cell cycle.

Epigenetic alterations associated with dexamethasone sodium phosphate through DNMT and TET in RPE cells.

Purpose: To elucidate the mechanism behind epigenetic alteration associated with dexamethasone (DEX) sodium phosphate treatment. Methods: We performed enzyme-linked immunosorbent assay to quantify changes in global DNA methylation and hydroxymethylation, quantitative real-time PCR (qRT-PCR) of the DNA methylation- and hydroxymethylation-related gene, in vitro DNA methyltransferase (DNMT) enzymatic activity assays with purified DNMTs, and DNA hydroxymethylation pattern with super-resolution imaging. Results: We identified global DNA hypomethylation and hyper-hydroxymethylation upon DEX treatment, associated with aberrant mRNA expression levels of DNMT and ten-eleven translocation (TET) proteins. Additionally, DEX exposure could directly hinder DNMT activities. Conclusions: We showed that DEX-induced epigenetic alterations are linked to aberrant DNMT and TET expression, potentially through an essential role of DNMT.

Colorimetric Detection of Organophosphate Pesticides Based on Acetylcholinesterase and Cysteamine Capped Gold Nanoparticles as Nanozyme.

Organophosphates (OPs) are neurotoxic agents also used as pesticides that can permanently block the active site of the acetylcholinesterase (AChE). A robust and sensitive detection system of OPs utilising the enzyme mimic potential of the cysteamine capped gold nanoparticles (C-AuNPs) was developed. The detection assay was performed by stepwise addition of AChE, parathion ethyl (PE)-a candidate OP, acetylcholine chloride (ACh), C-AuNPs, and 3, 3', 5, 5'-tetramethylbenzidine (TMB) in the buffer solution. The whole sensing protocol completes in 30-40 min, including both incubations. The Transmission Electron Microscopy (TEM) results indicated that the NPs are spherical and have an average size of 13.24 nm. The monomers of C-AuNPs exhibited intense catalytic activity (nanozyme) for the oxidization of TMB, revealed by the production of instant blue colour and confirmed by a sharp peak at 652 nm. The proposed biosensor's detection limit and linear ranges were 5.8 ng·mL-1 and 11.6-92.8 ng·mL-1, respectively, for PE. The results strongly advocate that the suggested facile colorimetric biosensor may provide an excellent platform for on-site monitoring of OPs.

Multi and transgenerational epigenetic effects of di-(2-ethylhexyl) phthalate (DEHP) in liver.

Di-(2-ethylhexyl) phthalate (DEHP), a ubiquitous industrial pollutant, is a known endocrine disrupter implicated in metabolic diseases. Prenatal DEHP exposure promotes epigenetic multi- and transgenerational inheritance of adult onset disease in subsequent generations (F1-F3). However, the epigenetic toxicity is less understood in the liver. In this study, CD-1 mice were prenatally exposed to 20 µg/kg/day, 200 µg/kg/day, 500 mg/kg/day, or 750 mg/kg/day DEHP from gestational day (GD) 10.5 until birth of pups. Following prenatal exposure, the multigenerational and transgenerational effects of mRNA expression of epigenetic regulators were evaluated in F1, F2, and F3 generation mouse livers at postnatal days (PNDs) 8 and 60. Results showed that DEHP exposed mice livers exhibited significant changes in global DNA methylation levels in all three generations, with the effect being different in F2 after high dosage exposure. Histopathology indicated that DEHP exposure could induce mild damage in F1 livers. The expression levels of DNA methyltransferase 1 (Dnmt1) were significantly changed in both the F1 and F2 generations at PND 8, suggesting that maintenance Dnmt1 plays a major role in the multigenerational effect that occur in the early developmental stages. Additionally, DEHP exposure caused significant changes in ten-eleven translocation methylcytosine (Tet) dioxygenases encoding Tet1 expression in all three generations and Tet2 expression in F3 at PND 60, implicating their contributions in inducing both multi- and transgenerational effects after DEHP exposure in mouse liver. Overall, our results establish that prenatal and ancestral DEHP exposure are critical for epigenetic regulation of DNA methylation in female mouse livers.

Laser Shock Tuning Dynamic Interlayer Coupling in Graphene-Boron Nitride Moiré Superlattices.

In the emergence of graphene and many two-dimensional (2D) materials, the most exciting applications come from stacking them into 3D devices, promising many excellent possibilities for neoteric electronics and optoelectronics. Layers of semiconductors, insulators, and conductors can be stacked to form van der Waals heterostructures, after the weak bonds formed between the layers. However, the interlayer coupling in these heterostructures is usually hard to modulate, resulting in difficulty to realize their emerging optical or electronic properties. Especially, the relationship between interlayer distance and interlayer coupling remains to be investigated, due to the lack of effective technology. In this work, we have used laser shocking to controllably tune the interlayer distance between graphene (Gr) and boron nitride (BN) in the Gr/BN/Gr heterostructures and the strains in the 2D heterolayers, providing a simple and effective way to modify their optic and electronic properties. After lase shocking, the reduction of interlayer distance is calculated by molecular dynamics (MD) simulation. Some atoms in Gr or BN are out-of-plane as well. In Raman measurements, the G peak in the heterostructure shows a red-shifted trend after laser shocking, indicating the strong phonon coupling in the interlayer. Moreover, the larger transparency after laser shocking also verifies the stronger photon coupling in the heterostructure. To investigate the effects of the interlayer coupling of heterostructure on its out-of-plane electronic behavior, we have investigated the electronic tunneling behavior. The heterostructure after laser shock reveals a lager tunneling current and lower tunneling threshold, proving an unexpected better electrical property. From DFT calculations, laser shocking can modulate the band gap structure of graphene in Gr/BN/Gr heterostructures; therefore, the heterostructures can be implemented as a unique photonic platform to modulate the emission characters of the anchored CdSe/ZnS core-shell quantum dots. Remarkably, the effective laser shocking method is also applicable to various otherwise noninteracting 2D materials, resulting in many new phenomena, which will lead science and technology to unexplored territories.

Magnetic Focus Lateral Flow Sensor for Detection of Cervical Cancer Biomarkers.

We report on a magnetic focus lateral flow biosensor (mLFS) for ultrasensitive detection of protein biomarkers in a practical format. With valosin-containing protein as a target protein, we show that the developed mLFS concept could detect as low as 25 fg/mL with magnetic focus to enhance target capture efficiency to deliver a 106-fold improvement in sensitivity compared to that of conventional lateral flow (LF) systems. The conceptualized strategy utilizes a simple magnet placed beneath the three-dimensional printed LF device to concentrate the targets at the signal zone without any additional instrumentation. In addition, protein mixtures extracted from the tissue of cervical cancer patients was also utilized to validate the sensor. To investigate the effect of magnetic focus on sensitivity, surface-enhanced Raman spectroscopy and dark-field imaging was utilized to characterize the distribution and movement of Fe3O4 core-Au shell nanoprobes in a model LF strip. Our experiments show that the magnetic focus results in an increased interaction time between the magnetic probe-labeled targets and the capture antibody, yielding a higher capture efficiency, allowing for ultrasensitive detection of the target not possible before with LF. The proposed mLFS can be utilized to detect a range of trace protein biomarkers for early diagnosis and can be combined with diverse pretreatment and signal amplification steps to query complex samples.

Prenatal and ancestral exposure to di(2-ethylhexyl) phthalate alters gene expression and DNA methylation in mouse ovaries.

Di(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer and known endocrine disrupting chemical, which causes transgenerational reproductive toxicity in female rodents. However, the mechanisms of action underlying the transgenerational toxicity of DEHP are not understood. Therefore, this study determined the effects of prenatal and ancestral DEHP exposure on various ovarian pathways in the F1, F2, and F3 generations of mice. Pregnant CD-1 dams were orally exposed to corn oil (vehicle control) or DEHP (20 µg/kg/day-750 mg/kg/day) from gestation day 10.5 until birth. At postnatal day 21 for all generations, ovaries were removed for gene expression analysis of various ovarian pathways and for 5-methyl cytosine (5-mC) quantification. In the F1 generation, prenatal DEHP exposure disrupted the expression of cell cycle regulators, the expression of peroxisome-proliferator activating receptors, and the percentage of 5-mC compared to control. In the F2 generation, exposure to DEHP decreased the expression of steroidogenic enzymes, apoptosis factors, and ten-eleven translocation compared to controls. It also dysregulated the expression of phosphoinositide 3-kinase (PI3K) factors. In the F3 generation, ancestral DEHP exposure decreased the expression of steroidogenic enzymes, PI3K factors, cell cycle regulators, apoptosis factors, Esr2, DNA methylation mediators, and the percentage of 5-mC compared to controls. Overall, the data show that prenatal and ancestral DEHP exposure greatly suppress gene expression of pathways required for folliculogenesis and steroidogenesis in the ovary in a transgenerational manner and that gene expression may be influenced by DNA methylation. These results provide insight into some of the mechanisms of DEHP-mediated toxicity in the ovary across generations.

Multiplex single-cell quantification of rare RNA transcripts from protoplasts in a model plant system.

Here we demonstrate multiplex and simultaneous detection of four different rare RNA species from plant, Arabidopsis thaliana, using surface-enhanced Raman spectroscopy (SERS) and gold nanoprobes at single-cell resolution. We show the applicability of nanoparticle-based Raman spectroscopic sensor to study intracellular RNA copies. First, we demonstrate that gold-nanoparticles decorated with Raman probes and carrying specific nucleic acid probe sequences can be uptaken by the protoplasts. We confirm the internalization of gold nanoprobes by transmission electron microscopy, inductively-coupled plasma-mass spectrometry and fluorescence imaging. Second, we show the utility of a SERS platform to monitor individual alternatively spliced (AS) variants and miRNA copies within single cells. Finally, the distinctive spectral features of Raman-active dyes were exploited for multiplex analysis of AtPTB2, AtDCL2, miR156a and miR172a. Furthermore, single-cell studies were validated by in vitro quantification and evaluation of nanotoxicity of gold probes. Raman tag functionalized gold nanosensors yielded an approach for the tracking of rare RNAs within the protoplasts. The SERS-based approach for quantification of RNAs has the capability to be a highly sensitive, accurate and discerning method for single-cell studies including AS variants quantification and rare miRNA detection in specific plant species.

Alpha Lead Oxide (α-PbO): A New 2D Material with Visible Light Sensitivity.

Even though transition metal dichalcogenides (TMDCs) are deemed to be novel photonic and optoelectronic 2D materials, the visible band gap being often limited to monolayer, hampers their potential in niche applications due to fabrication challenges. Uncontrollable defects and degraded functionalities at elevated temperature and under extreme environments further restrict their prospects. To address such limitations, the discovery of a new 2D material, α-PbO is reported. Micromechanical as well as sonochemical exfoliation of 2D atomic sheets of α-PbO are demonstrated and its optical behavior is investigated. Spectroscopic investigations indicate layer dependent band gaps. In particular, even multilayered PbO sheets exhibit visible band gap > 2 eV (direct) which is rare among semiconducting 2D materials. The emission lifetime of multilayer PbO atomic sheets is 7 ns (dim light) as compared to the monolayer which gives 2.5 ns lifetime and an intense light. Density functional theory calculations of layer dependent band structure of α-PbO matches well with experimental results. Experimental findings suggest that PbO atomic sheets exhibit hydrophobic nature, thermal robustness, microwave stability, anti-corrosive behaviour and acid resistance. This new low-cost, abundant and robust 2D material is expected to find many applications in the fields of electronics, optoelectronics, sensors, photocatalysis and energy storage.

Impedimetric detection of bacteria by using a microfluidic chip and silver nanoparticle based signal enhancement.

The authors describe a method that can significantly improve the performance of impedimetric detection of bacteria. A multifunctional microfluidic chip was designed consisting of interdigitated microelectrodes and a micro-mixing zone with a Tesla structure. This maximizes the coating of bacterial surfaces with nanoparticles and results in improved impedimetric detection. The method was applied to the detection of Escherichia coli O157:H7 (E. coli). Silver enhancement was accomplished by coating E.coli with the cationic polymer diallyldimethylammonium chloride (PDDA) to form positively charged E. coli/PDDA complexes. Then, gold nanoparticles (AuNPs) were added, and the resulting E. coli/PDDA/AuNPs complexes were collected at interdigitated electrodes via positive dielectrophoresis (pDEP). A silver adduct was then formed on the E. coli/PDDA/AuNP complexes by using silver enhancement solutions and by using the AuNPs as catalysts. The combination of pDEP based capture and of using silver adducts reduces impedance by increasing the conductivity of the solution and the double layer capacitance around the microelectrodes. Impedance decreases linearly in the 2 × 103-2 × 105 cfu·mL-1 E. coli concentration range, with a 500 cfu·mL-1 detection limit. Egg shell wash samples and tap water spiked with E. coli were successfully used for validation, and this demonstrates the practical application of this method. Graphical abstract Schematic representation of the AuNP@Ag enhancement method integrated with multifunctional microfluidic chip platform for impedimetric quantitation of bacteria. The method significantly improves the performance of impedimetric detection of bacteria.

Current Technologies of Electrochemical Immunosensors: Perspective on Signal Amplification.

An electrochemical immunosensor employs antibodies as capture and detection means to produce electrical charges for the quantitative analysis of target molecules. This sensor type can be utilized as a miniaturized device for the detection of point-of-care testing (POCT). Achieving high-performance analysis regarding sensitivity has been one of the key issues with developing this type of biosensor system. Many modern nanotechnology efforts allowed for the development of innovative electrochemical biosensors with high sensitivity by employing various nanomaterials that facilitate the electron transfer and carrying capacity of signal tracers in combination with surface modification and bioconjugation techniques. In this review, we introduce novel nanomaterials (e.g., carbon nanotube, graphene, indium tin oxide, nanowire and metallic nanoparticles) in order to construct a high-performance electrode. Also, we describe how to increase the number of signal tracers by employing nanomaterials as carriers and making the polymeric enzyme complex associated with redox cycling for signal amplification. The pros and cons of each method are considered throughout this review. We expect that these reviewed strategies for signal enhancement will be applied to the next versions of lateral-flow paper chromatography and microfluidic immunosensor, which are considered the most practical POCT biosensor platforms.

Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage.

Nuclear functions including gene expression, DNA replication and genome maintenance intimately rely on dynamic changes in chromatin organization. The movements of chromatin fibers might play important roles in the regulation of these fundamental processes, yet the mechanisms controlling chromatin mobility are poorly understood owing to methodological limitations for the assessment of chromatin movements. Here, we present a facile and quantitative technique that relies on photoactivation of GFP-tagged histones and paired-particle tracking to measure chromatin mobility in live cells. We validate the method by comparing live cells to ATP-depleted cells and show that chromatin movements in mammalian cells are predominantly energy dependent. We also find that chromatin diffusion decreases in response to DNA breaks induced by a genotoxic drug or by the ISceI meganuclease. Timecourse analysis after cell exposure to ionizing radiation indicates that the decrease in chromatin mobility is transient and precedes subsequent increased mobility. Future applications of the method in the DNA repair field and beyond are discussed.

Single molecule Raman spectroscopic assay to detect transgene from GM plants.

Substantial concerns have been raised for the safety of transgenics on human health and environment. Many organizations, consumer groups, and environmental agencies advocate for stringent regulations to avoid transgene products' contamination in food cycle or in nature. Here we demonstrate a novel approach using surface enhanced Raman spectroscopy (SERS) to detect and quantify transgene from GM plants. We show a highly sensitive and accurate quantification of transgene DNA from multiple transgenic lines of Arabidopsis. The assay allows us to detect and quantify the transgenes as low as 0.10 pg without need for PCR-amplification. This technology is relatively cheap, quick, simple, and suitable for detection at low target concentration.

An integrated microsystem with dielectrophoresis enrichment and impedance detection for detection of Escherichia coli.

An integrated microsystem device with matched interdigitated microelectrode chip was fabricated for enrichment and detection of Escherichia coli O157:H7. The microsystem has integrated with positive dielectrophoresis (pDEP) enrichment and in situ impedance detection, whose total volume is only 3.0 × 10-3 m3, and could provide impedance testing voltages of 0 ~ 10 V, detection frequencies of 1 KHz ~ 1 MHz, DEP excitation signals with amplitude of 0 ~ 10 Vpp and frequencies of 1KHz ~ 1 MHz, which fully meets the demands of pDEP enrichment and impedance detection for bacteria. The microfluidic chip with interdigitated microelectrodes was manufactured by microfabrication methods. The interdigital microelectrode array has sufficient contact area with a bacterial suspension to improve enrichment efficiency and detection sensitivity. Bacteria in the interdigital microelectrode area of the microfluidic chip were firstly captured and enriched by pDEP. Then, in situ impedance detection of the enriched bacteria was realized by switching test conditions. Using the self-assembly microsystem, a novel quantitative detection method was established and demonstrated to detect Escherichia coli O157:H7. Experimental results showed that the detection limits of Escherichia coli O157:H7 was 5 × 104 cfu mL-1, and testing time was only 6 min under the optimized detection voltage of 100 mV and frequency of 500 KHz. The method was successfully used to detect Escherichia coli O157:H7 in synthetic chicken synthetic samples.

Monitoring focal adhesion kinase phosphorylation dynamics in live cells.

Focal adhesion kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase essential for a diverse set of cellular functions. Current methods for monitoring FAK activity in response to an extracellular stimulus lack spatiotemporal resolution and/or the ability to perform multiplex detection. Here we report on a novel approach to monitor the real-time kinase phosphorylation activity of FAK in live single cells by fluorescence lifetime imaging.