Causal impact of DNA methylation on refracture in elderly individuals with osteoporosis - a prospective cohort study.

BACKGROUND: Osteoporotic vertebral compression fractures (OVCF) in the elderly increase refracture risk post-surgery, leading to higher mortality rates. Genome-wide association studies (GWAS) have identified susceptibility genes for osteoporosis, but the phenotypic variance explained by these genes has been limited, indicating the need to explore additional causal factors. Epigenetic modifications, such as DNA methylation, may influence osteoporosis and refracture risk. However, prospective cohorts for assessing epigenetic alterations in Chinese elderly patients are lacking. Here, we propose to conduct a prospective cohort study to investigate the causal network of DNA polymorphisms, DNA methylation, and environmental factors on the development of osteoporosis and the risk of refracture. METHODS: We will collect vertebral and peripheral blood from 500 elderly OVCF patients undergoing surgery, extract DNA, and generate whole genome genotype data and DNA methylation data. Observation indicators will be collected and combined with one-year follow-up data. A healthy control group will be selected from a natural population cohort. Epigenome-wide association studies (EWAS) of osteoporosis and bone mineral density will be conducted. Differential methylation analysis will compare candidate gene methylation patterns in patients with and without refracture. Multi-omics prediction models using genetic variants and DNA methylation sites will be built to predict OVCF risk. DISCUSSION: This study will be the first large-scale population-based study of osteoporosis and bone mineral density phenotypes based on genome-wide data, multi-time point methylation data, and phenotype data. By analyzing methylation changes related to osteoporosis and bone mineral density in OVCF patients, the study will explore the feasibility of DNA methylation in evaluating postoperative osteoporosis intervention effects. The findings may identify new molecular markers for effective anti-osteoporosis treatment and inform individualized prevention and treatment strategies. TRIAL REGISTRATION: chictr.org.cn ChiCTR2200065316, 02/11/2022.

Gravity-driven flow control in a fully integrated microfluidic cartridge for molecular point-of-care testing.

Molecular point-of-care testing (POCT) system is crucial for the timely prevention and control of infectious diseases. We recently proposed a gravity-driven microfluidic cartridge for molecular POCT detection, without the need for external sources or actuators, demonstrating the advantages in terms of the reduced cartridge size and low development costs. How to achieve precise control of liquid flow behavior is challenging for the gravity-driven cartridge. In this work, we explored the underlying mechanism of flow control in the cartridge and offered optimized solutions for our cartridge design to achieve precise control of dynamic flow rates and enhance pumping efficiency significantly. Through the computational fluid dynamics simulations, we demonstrated that adopting an asymptotic contraction chamber geometry design and a closed-loop air flow channel design with the cartridge inlet can facilitate stable laminar flow of the liquid in our microfluidic cartridge, enabling precise control of flow velocity. We further optimized the microchannel diameter and the contact angle of the liquid with the microchannel wall. The effectiveness of the optimized cartridge for POCT detection was well validated by the accurate detection of the human papillomavirus type 16 virus in the 120 clinical swab samples.

A sample-to-answer, quantitative real-time PCR system with low-cost, gravity-driven microfluidic cartridge for rapid detection of SARS-CoV-2, influenza A/B, and human papillomavirus 16/18.

The pandemic of coronavirus disease 2019 (COVID-19), due to the novel coronavirus (SARS-CoV-2), has created an unprecedented threat to the global health system, especially in resource-limited areas. This challenge shines a spotlight on the urgent need for a point-of-care (POC) quantitative real-time PCR (qPCR) test for sensitive and rapid diagnosis of viral infections. In a POC system, a closed, single-use, microfluidic cartridge is commonly utilized for integration of nucleic acid preparation, PCR amplification and florescence detection. But, most current cartridge systems often involve complicated nucleic acid extraction via active pumping that relies on cumbersome external hardware, causing increases in system complexity and cost. In this work, we demonstrate a gravity-driven cartridge design for an integrated viral RNA/DNA diagnostic test that does not require auxiliary hardware for fluid pumping due to adopted extraction-free amplification. This microfluidic cartridge only contains two reaction chambers for nucleic acid lysis and amplification respectively, enabling a fast qPCR test in less than 30 min. This gravity-driven pumping strategy can help simplify and minimize the microfluidic cartridge, thus enabling high-throughput (up to 12 test cartridges per test) molecular detection via a small cartridge readout system. Thus, this work addresses the scalability limitation of POC molecular testing and can be run in any settings. We verified the analytical sensitivity and specificity of the cartridge testing for respiratory pathogens and sexually transmitted diseases using SARS-CoV-2, influenza A/B RNA samples, and human papillomavirus 16/18 DNA samples. Our cartridge system exhibited a comparable detection performance to the current gold standard qPCR instrument ABI 7500. Moreover, our system showed very high diagnostic accuracy for viral RNA/DNA detection that was well validated by ROC curve analysis. The sample-to-answer molecular testing system reported in this work has the advantages of simplicity, rapidity, and low cost, making it highly promising for prevention and control of infectious diseases in poor-resource areas.

Multiple chemiluminescence immunoassay detection of the concentration ratio of glycosylated hemoglobin A1c to total hemoglobin in whole blood samples.

The concentration ratio of glycosylated hemoglobin A1c (HbA1c) to total hemoglobin (Hb) has long been used to accurately determine stagewise diabetes because this parameter represents a reliable and accurate biomarker of mean 90-day blood glucose values. In this paper, we report a time-resolved chemiluminescence assay that can detect both Hb and HbA1c. For the determination of Hb, the interaction of heme in Hb with H2O2 in NaOH solution was performed to generate a chemiluminescence peak. HbA1c was detected using a sandwich immunoassay based on an acridine ester-labeling method using the same Hb chemiluminescence trigger system. The results showed that the repeatability %CV of the proposed method for multiple detections of HbA1c and Hb ranged from 1.22 to 2.21%, with a median value of 1.73%, while the within-site reproducibility %CV ranged from 2.13 to 3.27%, with a median value of 2.81%. Compared with the conventional HPLC method (BIO-RAD D10 system), the correlation coefficient was 0.9959. In conclusion, a time-resolved multiple chemiluminescence immunoassay biosensor for HbA1c/Hb detection was established, and the method has excellent reproducibility and accuracy, thus demonstrating great potential for clinical application.

AutoCell Systematic Evolution of Ligands by Exponential Enrichment: The Software Designed and Developed for the Automated Screening System of Nucleic Acid Aptamers.

Aptamers are a new kind of nano-probes for bioassays and drug delivery, etc. In this paper, software has been developed as an automatic control center for the automated aptamer selecting system which realized the high integration of aptamer selection, data acquisition and processing. This software, applied in windows system, is developed by C# with the Microsoft Visual Studio 2015 integrated developing environment and the database used in this software is implemented using open source relational database MYSQL. According to the requirement analysis, this software realized various important necessary functions including feasible experiment design, auto-control of the hardware, real time process monitoring and efficient data management which perfectly satisfies the users' demands. During the actual experiment operation, this software worked smoothly and assumed stable serial port communication between it and the hardware, meanwhile, the interaction between the software and MYSQL remained good stability. As a consequence, it is practical and reasonable to apply this software to the automated aptamer selecting system for research.

Molecular dynamics discrimination of the conformational states of calmodulin through solid-state nanopores.

As a type of biological macromolecule, the conformation of proteins dynamically changes in a solution, which often results in a change in their function. However, traditional biological assays have significant drawbacks in detecting the conformation properties of proteins. Alternatively, nanopores have potential advantages in this area, which can detect protein in high throughput and without labelling. Herein, we investigated the translocation of calmodulins through silicon nitride nanopores using molecular dynamics (MD) simulation. Initially, the calmodulins were fixed in the nanopore. Distinguished blocked ionic currents were obtained between the two forms of calmodulin. Next, in the translocation simulations, a prominent difference in time resolution was easily found between the two states of calmodulin by using the appropriate voltage and comparable size of pore to protein, rp/rg→ 1, 4.5 nm (where rp is the protein radius and rg is the gyration radius). These simulations on the nanoscale are helpful for developing Ca2+-sensitive ion channels and nanodevices.

Design and Implementation of High-Throughput Magnetic Separation Module for Automated Nucleic Acid Detection System Based on Magnetic Nano-Beads.

With the higher and higher application level of medical technology, more and more genetic diseases have been diagnosed. Nucleic acid, as an important genetic material, has been found to have important functions in the storage and transmission of the genetic information in the replication and synthesis of proteins. As the first step in nucleic acid detection experiments, nucleic acid extraction performance is associated with the purity of target nucleic acid samples, which is very important for the downstream steps. In this paper, we employed the magnetic bead for extracting nucleic acids based on the platform of large liquid handling workstation and designed a matching magnetic separation module. It was shown that the temperature control block designed in this paper has reliable stability, high accuracy by using the incremental PID algorithm, with the control accuracy up to ±0.5 °C, and the control stabilization time is about 90 s, which can satisfy the experimental requirements. Besides, the average magnetic bead transfer rate of this module was further verified by mimicking the manual magnetic bead nucleic acid extraction process. The results proved that the module has an excellent performance with the average magnetic bead transfer rate greater than 95% and the magnetic bead transfer rate in each well greater than 90%, which could be consistent with the experimental indictors of nucleic acid extraction.

Design and Implementation of a High-Throughput Vibrating Module for Nucleic Acid Detection System.

The high-throughput nucleic acid detection system provides a good solution for detecting nucleic acids more safely, rapidly and accurately, which greatly improves the detection efficiency. Highthroughput nucleic acid detection mainly includes three steps: signal acquisition, signal amplification and signal processing. Therefore, obtaining the purified nucleic acid is the primary task of the nucleic acid detection, and the quality of the nucleic acid has a significant impact on results. In this paper, we employed the magnetic nanoparticle technology for extracting nucleic acids based on the platform of large liquid handling workstation and designed a matching vibrating module. The involved steps of core method, magnetic bead nucleic acid extraction technology, are mainly concerned with the cell lysis, nucleic acid binding, nucleic acid purification and magnetic particles elution. During the extraction process, specific temperature is required for the lysis and elution. It was shown that the temperature control part designed in this paper has the reliable stability, high accuracy by using the incremental proportion-integration-differentiation (PID) algorithm, with the control accuracy up to ±0.5 °C. The temperature regulating time is about 90 s, which can meet the experimental requirements. Besides, the vibrating uniformity of this module was further verified by protein concentration test, which proved that the module has the excellent performance and can be consistent with the experimental indictors of the nucleic acid extraction.

Mechanical Gripper Design and Force Analysis of Microplates for Automated High-Throughput Nucleic Acid Detection System.

In the automated high-throughput nucleic acid detection system, we need to grip and transfer consumables frequently when carrying out multichannel nucleic acid detection. In order to ensure the efficiency of experiments and solve problems of the deflection and drop when transferring microplates, we design a self-locking mechanical gripper which consists of a rotary positioning module and a gripping module. The absolute position encoder fixed on the top of the stepper motor can collect the position data of the mechanical gripper in real time and send them to the master control board based on STM32 for processing, which ensures the accuracy of the movement of the mechanical gripper. We used SolidWorks to build models of the mechanical gripper and different microplates, and we carried on finite element analysis of microplates to find the suitable gripping position. Through the force analysis, we obtained the pressure distribution and the deformation of different microplates, and defined the effective gripping areas, which is important to the grip and transfer of microplates.

One-Step Synthesis of DNA Templated Water-Soluble Au-Ag Bimetallic Nanoclusters for Ratiometric Fluorescence Detection of DNA.

DNA strands have been used as templates to form different sized silver nanoclusters. Although DNA templated silver nanoclusters (NCs) probes which show outstanding fluorescence properties have been widely applied in chemical sensing and cellular imaging, synthesis of DNA template Au-Ag bimetallic nanoclusters remains a challenge. A facile one-step synthesis method was thus developed in this study to form Au-Ag NCs using C4-ATAT-C4 as template. C4-ATAT-C4 acted as a stabilizer for preventing aggregation of the Au-Ag NCs. The obtained Au-Ag NCs stabilized by C4-ATAT-C4 showed bright fluorescence and high stability. A series of experiments showed that temperature, citrate-citric acid buffer, sequence and concentration of DNA played an important role in the synthesis of fluorescent Au-Ag NCs. In addition, a ratiometric fluorescence probe was designed through combining the nucleotide sequence (C4-ATAT-C4) and hybridization sequence in the presence of the double-strand-chelating dye Super SYBR Green (SG). The combination of prepared Au-Ag NCs, Super SG, and perfectly matched DNA emitted fluorescence at 500 and 590 nm, respectively, when the composite was excited at 290 nm. A gastric cancer gene was also selectively detected by our developed ratiometric fluorescence probe.

Environmental effect on surface immobilized biological molecules.

Our recent sum frequency generation (SFG) vibrational spectroscopic experiment ( J. Phys. Chem. B 2014 , 118 , 2904 - 2912 ) showed that immobilized antimicrobial peptide cecropin P1 (cCP1) on a self-assembled monolayer (SAM) surface via N-terminus exhibited significantly different conformational and/or orientational behaviors when exposed to pure water vs a 50% (v/v) 2,2,2-trifluoroethanol (TFE)/water mixture. Meanwhile, our recent molecular dynamics (MD) simulations ( J. Phys. Chem. B 2014 , 118 , 5670 - 5680 ) further revealed that the immobilized cCP1 via N-terminus in pure water largely adopts an overall bent structure lying down on the SAM surface, consistent with the SFG observation. Here, MD simulations were performed on the immobilized cCP1 on a SAM surface via N-terminus while in contact with a 50% (v/v) TFE/water mixture to further investigate the effects of environment (water vs TFE/water mixture) on the interfacial structure and orientation of immobilized peptide. The simulation results demonstrated that the immobilized cCP1 on the SAM surface via the N-terminus with two different starting states with different orientations and conformations, when exposed to a 50% (v/v) TFE/water mixture, was eventually able to maintain a linear α-helical structure, standing upright on the SAM surface. Taken with the corresponding SFG observation, our simulation results indicate that the conformational behavior of the immobilized peptide is mediated by the local hydrophobic environments resulting from the TFE aggregation around the peptide. Such knowledge can be used to regulate the surface conformation and functionality of immobilized peptides via changing surrounding chemical environments (e.g., TFE cosolvent), which is important for the microbial detection and killing based on surface-immobilized antimicrobial peptides.

Molecular structures of C- and N-terminus cysteine modified cecropin P1 chemically immobilized onto maleimide-terminated self-assembled monolayers investigated by molecular dynamics simulation.

Biosensors using peptides or proteins chemically immobilized on surfaces have many advantages such as better sensitivity, improved stability, and longer shelf life compared to those prepared using physically adsorbed biomolecules. Chemical immobilization can better control the interfacial conformation and orientation of peptides and proteins, leading to better activity of these biomolecules. In this research, molecular dynamics (MD) simulations were employed to systematically investigate the structure and dynamics of surface-tethered antimicrobial peptide cecropin P1 (CP1) modified with a cysteine residue at the C- (CP1c) or N-terminus (cCP1). Such CP1c and cCP1 molecules were chemically immobilized onto a silane-EG4-maleimide self-assembled monolayer (SAM) surface by forming a thio-ether bond between the cysteine group in CP1c or cCP1 and the surface maleimide group. The simulation results showed that the immobilized cCP1 (via the N-terminus) tends to bend and gradually lie down onto the SAM surface, due to the large structural fluctuation of the C-terminus induced by unfavorable interactions between the hydrophobic C-terminal residues and water. Differently, the tethered CP1c (via the C-terminus) more or less stands up on the surface, only tilting slightly even after 60 ns. The simulation results can be well correlated to the recent experimental results obtained from sum frequency generation (SFG) vibrational spectroscopic study. The current simulation data provide more atomic level details on how the hydrophobicity difference in the C-terminus and N-terminus of the amphiphilic peptide can lead to different structures of the same peptide tethered to the surface via different termini. This knowledge can be used to rationally design chemically immobilized peptides to achieve desired structure and functionality.

Different interfacial behaviors of peptides chemically immobilized on surfaces with different linker lengths and via different termini.

Molecular structures such as conformation and orientation are crucial in determining the activity of peptides immobilized to solid supports. In this study, sum frequency generation (SFG) vibrational spectroscopy was applied to investigate such structures of peptides immobilized on self-assembled monolayers (SAMs). Here cysteine-modified antimicrobial peptide cecropin P1 (CP1) was chemically immobilized onto SAM with a maleimide terminal group. Two important characteristics, length of the poly(ethylene glycol) (PEG) segment in the SAM and location of the cysteine residue in the peptide, were examined using SFG spectroscopy to determine the effect of each on surface immobilization as well as peptide secondary structure and its orientation in the immobilized state. Results have shown that while each length of PEG chain studied promotes chemical immobilization of the target peptide and prevents nonspecific adsorption, CP1 immobilized on long-chain (PEG2k) maleimide SAMs shows random coil structure in water, whereas CP1 demonstrates α-helical structure when immobilized on short-chain (with four ethylene glycol units - (EG4)) maleimide SAMs. Placement of the cysteine residue at the C-terminus promotes the formation of α-helical structure of CP1 with a single orientation when tethered to EG4 maleimide SAM surfaces. In contrast, immobilization via the N-terminal cysteine of CP1 results in a random coil or lying-down helical structure. The bacteria capturing/killing capability was tested, showing that the surface-immobilized CP1 molecules via C- and N- terminal cysteine exhibit only slight difference, even though they have different secondary structures and orientations.

PEGylated reduced graphene oxide as a superior ssRNA delivery system.

Single stranded ribonucleic acid (ssRNA) acts as a probe, antisense (AS), miRNA analog and inhibitor, and is promising for gene therapy and molecular diagnosis. However, free ssRNA exhibits poor cellular uptake due to its negative charges, and enzyme instability, which have largely limited the practical applications of ssRNA in biomedicine. To address these issues, we have developed a PEGylated reduced graphene oxide (PEG-RGO) nanovector for efficient delivery of ssRNA. We have demonstrated that PEG-RGO exhibits superior ssRNA loading and delivery capability, compared to the widely studied PEGylated graphene oxide (PEG-GO). Computational simulation further suggested that PEG-RGO binds ssRNA much stronger than PEG-GO, consistent with the experimental results. These results will have implications in designing RGO-based biocompatible and efficient ssRNA delivery systems.

Molecular dynamics simulations of end-tethered single-stranded DNA probes on a silica surface.

The structure of DNA molecules tethered to surfaces may significantly affect the efficiency of hybridization on the DNA microarray. Understanding the structure of single-stranded DNA (ssDNA) tethered to surfaces is critical for applying the molecular recognition function of DNA microarrays. Although a number of experimental methods have been applied to determine the structure of the DNA probe on surfaces, they can not provide enough information on the dynamical behavior of the ssDNAs on surfaces. Herein, we investigated the dynamics and interaction of seven DNA probes tethered on a silica surface by a molecular dynamics simulation. From the simulation results, we examined the structure and dynamics of the ssDNAs, by calculating the root-mean-square derivations, the tilt angles, the radius of gyration, and the distances of the neighboring ssDNAs. The data obtained from our simulation suggests the packing density has a significant effect on the overall structure and molecular orientation change of surface-tethered ssDNAs, which is complementary to the recent experimental reports. Our simulation provided a structural insight, which is helpful to better understand the behavior of ssDNA on surfaces and optimize the design of DNA microarray.

Effect of single central base pair mismatch on the conformation and stability of surface immobilized DNA: molecular dynamics studies.

We combine molecular dynamics simulations and relative free energy calculation to investigate how single central base mismatch influence the conformation and stability of surface immobilized DNA. For comparison purpose, molecular dynamics simulations have been performed on a central mismatched duplex and the corresponding perfectly matched duplex, respectively. Simulations have been conducted with the generalized Born model. The relative free energy differences of the two systems suggest the single base-pair mismatch may lower the stability of DNA as expectation. The result obtained from the structural analysis shows the average conformations of the two DNA duplexes resemble the B-form, which suggests the central base pair mismatch has little effect on the overall conformation of DNA. Furthermore, our result shows the fluctuation of the perfectly matched duplex is obviously larger than that of the central base pair mismatched duplex. This study gives us a new atomic-level insight into DNA probe-target interaction on the surface. The results presented here will also aid in the design of DNA microarrays for single nucleotide polymorphisms screening.

Preparation of SiO2/(PMMA/Fe3O4) from monolayer linolenic acid modified Fe3O4 nanoparticles via miniemulsion polymerization.

SiO2/(PMMA/Fe3O4) composite particles were prepared from linolenic acid (LA) instead of oleic acid (OA) modified Fe3O4 nanoparticles by miniemulsion polymerization. LA has three unsaturated double bonds with which it can polymerizate more easily than OA. And coating Fe3O4 with polymethyl methacrylate (PMMA) polymer beforehand can prevent magnetic nanoparticles from the aggregation that usually comes from the increasing of ionic strength during the hydrolyzation of tetraethoxysilane (TEOS) by the steric hindrance. Finally, the resulting PMMA/Fe3O4 nanoparticles were coated with silica, forming SiO2/(PMMA/Fe3O4) core-shell structure particles. The sizes of nanoparticles with core-shell structure were in the range from 300 to 600 nm. The nanoparticles were spherical particles and had consistent size. The result of magnetic measurement showed that the composite particles had superparamagnetic property.