Real-time Probe Research Articles

Glutathione-Responsive Multifunctional "Trojan Horse" Nanogel as a Nanotheranostic for Combined Chemotherapy and Photodynamic Anticancer Therapy.

It remains a great challenge to design a multifunctional and robust nanoplatform for stimuli-responsive drug delivery toward a lesion, which tactfully integrates multiple molecules with therapeutic and diagnostic characteristics. Herein, we reported a facile and ingenious cross-linked nanogel (DSA) based on the chemical cross-link of drugs as a straightforward strategy to overcome the instability of the assembly. In DSA, doxorubicin (DOX) and 5-aminolevulinic acid (ALA) were cross-linked with a disulfide linker for realizing synergistic anticancer therapy. The stability of DSA was adjusted via balancing the hydrophobic/hydrophilic property with hydrophilic NH2-PEG1k. After regulating the coordination of the DOX part and ALA moiety, the drug-loaded nanogel exhibited superior chemotherapeutic efficacies. Additionally, the DSA could selectively biosynthesize fluorescent protoporphyrin IX (PpIX) in tumor cells, which could be applied for a real-time imaging probe of accurate cancer diagnosis. Besides, the in situ synthesized PpIX in mitochondria could serve as a photosensitizer to convert oxygen into toxic reactive oxygen species under a near infrared ray at 660 nm irradiation, leading to an excellent tumor-killing efficacy. This work proposed a unique strategy for designing a series of prodrug nanogels as a universal drug delivery platform for realizing precise disease therapy and diagnostics.

In-situ and real-time probing cellulase biosensor formation and its interaction with lignosulfonate in varied media

It is a challenge to interpret the signals obtained by Quartz Crystal Microbalance (QCM) and Surface Plasmon Resonance (SPR) accompanied with in the significant changes of the density and/or refractivity of media. The work presented herein focuses on demonstrating a universal in-situ and real-time monitoring methodology for ultrathin film formation on gold chips in varied media. Biomimetic model cellulase/enzyme film was immobilized in-situ on gold surface of sensors, followed by revealing its interaction with lignosulphonate in the media changed from ethanol-based to aqueous ones. Combining the techniques of Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and Multi-parametric Surface Plasmon Resonance (MP-SPR) along with respective software and models enabled us to precisely characterize the thickness, viscous and shear elastic moduli, and the coupled solvent for individual layer formed over the entire process, which are unachievable with conventional methods. The methodology provides real-time monitoring of filming process and offers us an in-situ qualify control approach to the biomimetic model films formed on different substrates including gold chip.

Noninvasive Fetal Sex Determination by Real-Time PCR and TaqMan Probes.

Noninvasive fetal sex determination by analyzing Y chromosome-specific sequences is very useful in the management of cases related to sex-linked genetic diseases. The aim of this study was to establish a non-invasive fetal sex determination test using Real-Time PCR and specific probes. The study was a prospective observational cohort study conducted from August 2018 to September 2019. Venous blood samples were collected from 25 Iranian pregnant women at weeks 7 to 25 of gestation. Cell-free DNA (cfDNA) was isolated from the plasma of samples and fetal sex was determined by SRY gene analysis using the Real-Time PCR technique. In the absence of SRY detection, the presence of fetal DNA was investigated using cfDNA treated with BstUI enzyme and PCR for the epigenetic marker RASSF1A. Of the total samples analyzed, 48% were male and 52% female. The RASSF1A assay performed on SRY negative cases also confirmed the presence of cell-free fetal DNA. Genotype results were in full agreement with neonate gender, and the accuracy of noninvasive fetal sex determination was 100%. Fetal sex determination using the strategy applied in this study is noninvasive and highly accurate and can be exploited in the management of sex-linked genetic diseases.

Simultaneously prediction of sheep and goat carcass composition and body fat depots using in vivo ultrasound measurements and live weight

The present study established multiple linear regression models using two ultrasound in vivo measurements (at lumbar and sternal regions, with different real-time ultrasonography machines and probes) and live weight, to predict simultaneously carcass composition and body fat depots of different breeds of sheep and goat. This study is important for the small ruminant industry, considering the feasibility of using the ultrasound methodology in field conditions, as well as an online system of the carcass evaluation. The multiple linear regression models were obtained by selecting the best subset of variables between using the in vivo measurements (raw variables), their second degree and interactions, evaluated in terms of prediction performance using cross-validation “K-folds” and validated by a test group. Overall, high accuracy (adj R2) was obtained from the linear relationship between predicted and experimental values of the group test for each of the nine dependent variables, with values varying between adj R2 0.88 and 0.98.

Real-time nanodiamond thermometry probing in vivo thermogenic responses.

Real-time temperature monitoring inside living organisms provides a direct measure of their biological activities. However, it is challenging to reduce the size of biocompatible thermometers down to submicrometers, despite their potential applications for the thermal imaging of subtissue structures with single-cell resolution. Here, using quantum nanothermometers based on optically accessible electron spins in nanodiamonds, we demonstrate in vivo real-time temperature monitoring inside Caenorhabditis elegans worms. We developed a microscope system that integrates a quick-docking sample chamber, particle tracking, and an error correction filter for temperature monitoring of mobile nanodiamonds inside live adult worms with a precision of ±0.22°C. With this system, we determined temperature increases based on the worms' thermogenic responses during the chemical stimuli of mitochondrial uncouplers. Our technique demonstrates the submicrometer localization of temperature information in living animals and direct identification of their pharmacological thermogenesis, which may allow for quantification of their biological activities based on temperature.

Association of miR-196a2 and miR-149 single-nucleotide polymorphisms with atherosclerotic ischemic stroke susceptibility

BackgroundBeside common risk factors for stroke such as diabetes and hypertension, single-nucleotide variants occurring within micro RNA genes have been identified as susceptibility loci for ischemic stroke risk.ObjectivesInvestigate the possible association of two variants in pre miRNA sequences, rs11614913 within miR-196a2 C > T and rs2292832 within miR-149 T > C, with ischemic stroke.Subjects and methodsOne hundred ischemic stroke patients and 100 age and sex-matched controls having > 1 risk factor for atherosclerosis were enrolled in a case-control study. Degree of atherosclerosis was assessed using ultrasonography. Micro RNA variants were assessed by real-time PCR TaqMan probe assay.ResultsThe TT genotype and T allele frequencies of miR-196a2 C > T were protective against ischemic stroke (OR 0.168, P 0.001; OR 0.482, P < 0.001 respectively). While among miR-149 T > C variants, CC genotype was associated with increased risk by threefold (OR 3.061, P 0.005) and C allele was associated with about 1.9 fold risk of stroke (OR 1.909, P 0.002). Haplotypes analysis revealed miR-196a2T/-149 T allele combination was significantly lower among stroke patients than the controls (P < 0.001) with a protective effect (OR 0.196, 95% CI 0.083-0.466).ConclusionA decrease in the incidence of ischemic stroke is associated with miR-196a2 TT genotype and T allele and increases in the likelihood risk of ischemic stroke are associated with CC genotype and C allele within miR-149, and the two miRNAs under study are closely associated with vascular damage responses.

Optimization of extracorporeal membrane oxygenation therapy using near-infrared spectroscopy to assess changes in peripheral circulation: A pilot study.

Near-infrared spectroscopy (NIRS) has been proposed as a noninvasive modality for detecting complications in patients undergoing extracorporeal membrane oxygenation (ECMO), and it can simultaneously reveal the global circulatory status of these patients. We optimized ECMO therapy on the basis of real-time peripheral NIRS probing. Three patients underwent venoarterial (VA) ECMO and one patient underwent venovenous (VV) ECMO. All patients received peripheral ECMO cannulation with routine distal perfusion catheter placement. We designed an experimental protocol to adjust ECMO blood flow over 1 hour. Hemodynamic responses were measured using NIRS devices attached to the calf at approximately 60% of the distance from the ankle to the knee. HbO2 levels change substantially with adjustments in ECMO flow, and they are more sensitive than HHb levels and the tissue saturation index (TSI) are. NIRS for optimizing ECMO therapy may be reliable for monitoring global circulatory status.

Mitochondria targeted redox GFP reveals time and dose dependent onset and progression of mitochondrial oxidation with diverging cell death decisions during photodynamic therapy.

Photodynamic therapy (PDT) is a successful cancer treatment modality. In vitro, in vivo, and clinical studies with different photosensitizers reveal diverging cell fates, including apoptosis, necrosis, autophagy, and non-specific forms of cell death. The mode of action and efficacy of PDT is mediated through free radical generation and is highly dependent on diverse variables such as nature, dose, metabolism of photosensitizer, irradiation energy, and irradiation cycle. Discovery of newer photosensitizers and optimization of PDT approaches to achieve a clinically relevant form of cell death called apoptosis requires better in vitro real-time methods. Oxidative damage and mitochondrial permeabilization are critical signaling events involved in photodamage and apoptosis. Hence, mitochondrial damage detection is an appropriate target signaling for mechanistic evaluation of PDT. We report mitochondria-targeted redox GFP expressing cells as a sensitive system to test and validate important variables of PDT using the photosensitizer 5-Aminolevulinic acid (5-ALA) as a model. An independent FRET-based caspase sensor cell was also used to study the impact of the photosensitizer dosage and irradiation duration on the mode of cell death. The study reveals that the cancer cells expressing mt-roGFP are extremely sensitive to monitor mitochondrial oxidation induced by PDT. The extent of mitochondrial redox changes induced by PDT can be determined using these sensor cells by real-time image-based approaches. These approaches provide sufficient temporal resolution that is required to fine-tune and optimize the PDT conditions. The degree of oxidation of the probe is highly dependent on the dosage of photosensitizer and duration of light irradiation, which determines the nature of cell death. A real-time caspase sensor probe further confirmed that the caspase-dependent and caspase-independent nature of cell death is in high correlation with the extent of mitochondrial oxidation. A condition that triggers rapid and extreme mito-oxidation seems to favor necrosis, while delayed and slowly progressing redox changes contribute to caspase-dependent apoptosis. The study confirms that temporal analysis of mitochondrial oxidation is a reliable biomarker for fine-tuning PDT conditions to achieve the desired outcome. This can be achieved using stable cancer cell lines expressing mitochondria-targeted roGFP by ratiometric imaging.

THE RELEVANCE OF MEDICAL LABORATORY DIAGNOSIS IN CORONAVIRUS IN SUSPECTED HUMAN CASES

Coronaviruses are a large family of viruses that are known to cause illness ranging from the common cold to more severe diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome. Coronavirus disease (Covid-19) is an infectious disease caused by a new virus. It leads to respiratory illness with symptoms such as a cough, fever, and in more severe cases, difficulty breathing. One can be protected by washing hands regularly, avoiding touching your face, and avoiding close contact with individuals who are unhealthy. It is transmitted through contact with an infected individual when they cough or sneeze. It could be transmitted when an individual touches a surface or object that has the virus on it, then touches the eyes, nose, or mouth. Some laboratory test to diagnose covid-19 includes real-time RT-PCR primer and probe and CDC serology test. It is very necessary that covid-19 is diagnosed for accurate treatment and management.

Tracking and visualization of the sensing area for a tethered laparoscopic gamma probe

PurposeIn surgical oncology, complete cancer resection and lymph node identification are challenging due to the lack of reliable intraoperative visualization. Recently, endoscopic radio-guided cancer resection has been introduced where a novel tethered laparoscopic gamma detector can be used to determine the location of tracer activity, which can complement preoperative nuclear imaging data and endoscopic imaging. However, these probes do not clearly indicate where on the tissue surface the activity originates, making localization of pathological sites difficult and increasing the mental workload of the surgeons. Therefore, a robust real-time gamma probe tracking system integrated with augmented reality is proposed.MethodsA dual-pattern marker has been attached to the gamma probe, which combines chessboard vertices and circular dots for higher detection accuracy. Both patterns are detected simultaneously based on blob detection and the pixel intensity-based vertices detector and used to estimate the pose of the probe. Temporal information is incorporated into the framework to reduce tracking failure. Furthermore, we utilized the 3D point cloud generated from structure from motion to find the intersection between the probe axis and the tissue surface. When presented as an augmented image, this can provide visual feedback to the surgeons.ResultsThe method has been validated with ground truth probe pose data generated using the OptiTrack system. When detecting the orientation of the pose using circular dots and chessboard dots alone, the mean error obtained is 0.05^{circ } and 0.06^{circ }, respectively. As for the translation, the mean error for each pattern is 1.78 mm and 1.81 mm. The detection limits for pitch, roll and yaw are 360^{circ }, 360^{circ } and 8^{circ }–82^{circ }cup 188^{circ }–352^{circ } .ConclusionThe performance evaluation results show that this dual-pattern marker can provide high detection rates, as well as more accurate pose estimation and a larger workspace than the previously proposed hybrid markers. The augmented reality will be used to provide visual feedback to the surgeons on the location of the affected lymph nodes or tumor.

A fluorimetric water-soluble polymeric pH chemosensor for extremely acidic conditions: Live-cell and bacterial imaging application

In this study, a novel water-soluble difluoroboron dipyrromethene (BODIPY)-based polymeric fluorescent probe (P1) suitable for extremely acidic conditions is synthesized. Compared to conventional small-molecule-based organic pH sensors, the P1 polymeric probe exhibits excellent solubility and long-term stability in water, with instantaneous fluorimetric response to pH changes. This extreme pH response is attributed to protonation of tertiary amine groups of the BODIPY segment, which suppresses photo-induced electron transfer (PET) from the tertiary amine to the BODIPY skeleton. This mechanism is supported by density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations. The P1 also displays high selectivity, reversibility, and low cytotoxicity even at high concentration. Also, its fluorescence response in acidic pH is investigated for in vitro cell imaging and E. coli bacterial imaging experiments. Additionally, the feasibility of immobilizing the P1 on test paper highlights potential as a real-time probe for rapid color change via drop-casting of the pH solution (below pH 4). Through alternative exposure of hydrochloric acid/ammonia (acid/base) vapor, the P1 is also projected as an indirect ammonia vapor indicator.

Ultrafast magnetic dynamics in insulating YBa2Cu3O6.1 revealed by time resolved two-magnon Raman scattering

Measurement and control of magnetic order and correlations in real time is a rapidly developing scientific area relevant for magnetic memory and spintronics. In these experiments an ultrashort laser pulse (pump) is first absorbed by excitations carrying electric dipole moment. These then give their energy to the magnetic subsystem monitored by a time-resolved probe. A lot of progress has been made in investigations of ferromagnets but antiferromagnets are more challenging. Here, we introduce time-resolved two-magnon Raman scattering as a real time probe of magnetic correlations especially well-suited for antiferromagnets. Its application to the antiferromagnetic charge transfer insulator YBa2Cu3O6.1 revealed rapid demagnetization within 90 fs of photoexcitation. The relaxation back to thermal equilibrium is characterized by much slower timescales. We interpret these results in terms of slow relaxation of the charge sector and rapid equilibration of the magnetic sector to a prethermal state characterized by parameters that change slowly as the charge sector relaxes.

Molecular characterisation and phylogenetic analysis of Marek’s disease virus in Turkish layer chickens

ABSTRACT 1. There is no current data about the genotypes of Marek’s disease virus (MDV) in Turkish poultry flocks; hence, this study was performed to analyse CVI988/Rispens, turkey herpesvirus (HVT) vaccine viruses and MDV field viruses as well as to perform phylogenetic analysis of MDV in Turkish layer chickens. 2. In 2017 and 2018, a total of 602 spleen samples from 49 layer flocks were collected from the Marmara, West Black Sea and Aegean regions. DNA was extracted from the spleen samples and the samples were analysed by real-time PCR probe assay to detect CVI988/Rispens and HVT vaccine viruses and MDV field strains. Samples found positive for MDV by real-time PCR were subjected to PCR using the Meq gene primers for phylogenetic analysis. 3. Amongst 49 flocks, virulent MDV was detected in nine flocks. CVI988/Rispens and HVT vaccine strains were detected in 47 flocks and HVT in all 49 flocks. Splenomegaly, hepatomegaly and tumours in the oviduct were observed in chickens of affected flocks. Virulent MDV was detected in 120 out of 602 spleen samples. Sequencing and phylogenetic analyses showed that MDVs detected in this study were closely related to MDV strains from Italy, Poland, Saudi Arabia, Iraq, India and China but showed diversity with MDV strains from Egypt and Hungary. Multiple sequence analysis of the Meq protein revealed several point mutations in deduced amino acid sequences. Interestingly, CVI988/Rispens vaccine virus from China (AF493555) showed mutations at position 66 (G66R) and 71 (S66A) along with two other vaccine strains from China (GU354326.1) and Russia (EU032468.1), in comparison with the other vaccine strain CVI988/Rispens (DQ534538). The molecular analyses of the Meq gene suggested that Turkish field strains of MDV are in the class of virulent or very virulent pathotypes. 4. The results have shown that MDV still affects poultry health, and the phylogenetic and amino acid variation data obtained will help in vaccination and control strategies.

GoPrime: Development of an In Silico Framework to Predict the Performance of Real-Time PCR Primers and Probes Using Foot-and-Mouth Disease Virus as a Model.

Real-time PCR (rPCR) is a widely accepted diagnostic tool for the detection and quantification of nucleic acid targets. In order for these assays to achieve high sensitivity and specificity, primer and probe-template complementarity is essential; however, mismatches are often unavoidable and can result in false-negative results and errors in quantifying target sequences. Primer and probe sequences therefore require continual evaluation to ensure they remain fit for purpose. This paper describes the development of a linear model and associated computational tool (GoPrime) designed to predict the performance of rPCR primers and probes across multiple sequence data. Empirical data were generated using DNA oligonucleotides (n = 90) that systematically introduced variation in the primer and probe target regions of a diagnostic assay routinely used to detect foot-and-mouth disease virus (FMDV); an animal virus that exhibits a high degree of sequence variability. These assays revealed consistent impacts of patterns of substitutions in primer and probe-sites on rPCR cycle threshold (CT) and limit of detection (LOD). These data were used to populate GoPrime, which was subsequently used to predict rPCR results for DNA templates (n = 7) representing the natural sequence variability within FMDV. GoPrime was also applicable to other areas of the FMDV genome, with predictions for the likely targets of a FMDV-typing assay consistent with published experimental data. Although further work is required to improve these tools, including assessing the impact of primer-template mismatches in the reverse transcription step and the broader impact of mismatches for other assays, these data support the use of mathematical models for rapidly predicting the performance of rPCR primers and probes in silico.

Deep learning reconstruction of ultrashort pulses from 2D spatial intensity patterns recorded by an all-in-line system in a single-shot.

We propose a simple all-in-line single-shot scheme for diagnostics of ultrashort laser pulses, consisting of a multi-mode fiber, a nonlinear crystal and a camera. The system records a 2D spatial intensity pattern, from which the pulse shape (amplitude and phase) are recovered, through a fast Deep Learning algorithm. We explore this scheme in simulations and demonstrate the recovery of ultrashort pulses, robustness to noise in measurements and to inaccuracies in the parameters of the system components. Our technique mitigates the need for commonly used iterative optimization reconstruction methods, which are usually slow and hampered by the presence of noise. These features make our concept system advantageous for real time probing of ultrafast processes and noisy conditions. Moreover, this work exemplifies that using deep learning we can unlock new types of systems for pulse recovery.

Real-Time Monitoring of Colloidal Crystallization in Electrostatically-Levitated Drops.

Colloidal crystallization is analogous to the crystallization in bulk atomic systems in various aspects, which has been explored as a model system. However, a real-time probing of the phenomenon still remains challenging. Here, a levitation system for a study of colloidal crystallization is demonstrated. Colloidal particles in a levitated droplet are gradually concentrated by isotropic evaporation of water from the surface of the droplet, resulting in crystallization. The structural change of the colloidal array during crystallization is investigated by simultaneously measuring the volume and reflectance spectra of the droplet. The crystal nucleates from the surface of the droplet at which the volume fraction exceeds the threshold and then the growth proceeds. The crystal growth behavior depends on the initial concentrations of colloidal particles and salts which determine the overall direction of crystal growth and interparticle spacing, respectively. The results show that a levitating bulk droplet has a great potential as a tool for in situ investigation of colloidal crystallization.

Interface for Reproducible, Multishot Direct Analysis of Solid-Phase Microextraction Samples.

An enclosed interface that joins a direct analysis in real time (DART) probe, solid-phase microextraction (SPME) fiber, and the inlet of a high-resolution mass spectrometer is described. Unlike other systems to couple SPME sampling to ambient mass spectrometry, the interface is able to perform discrete analyses on different areas of a single SPME fiber device for up to three technical replicate measurements of one sampling event. Inlet flow speed and desorption temperature are optimized, and reproducibility is demonstrated between replicate analyses on the same derivatized SPME fiber and with sequential fiber sampling events, yielding analyte measurement center of variance (CV) from 3 to 6%. Conditioning is also performed with the enclosed DART. The interface is a straightforward addition to commercially available technologies, and machine diagrams for custom components operated with SPME/DART/MS equipment are included.

Association study of genetic polymorphisms in proteins involved in oseltamivir transport, metabolism, and interactions with adverse reactions in Mexican patients with acute respiratory diseases

Oseltamivir, a pro-drug, is the best option for treatment and chemoprophylaxis for influenza outbreaks. However, many patients treated with oseltamivir developed adverse reactions, including hypersensitivity, gastritis, and neurological symptoms. The aim of this study was to determine the adverse drug reactions (ADRs) in Mexican patients treated with oseltamivir and whether these ADRs are associated with SNPs of the genes involved in the metabolism, transport, and interactions of oseltamivir. This study recruited 310 Mexican patients with acute respiratory diseases and treated them with oseltamivir (75 mg/day for 5 days) because they were suspected to have influenza A/H1N1 virus infection. Clinical data were obtained from medical records and interviews. Genotyping was performed using real-time polymerase chain reaction and TaqMan probes. The association was assessed under genetic models with contingency tables and logistic regression analysis. Out of 310 patients, only 38 (12.25%) presented ADRs to oseltamivir: hypersensitivity (1.9%), gastritis (10%), and depression and anxiety (0.9%). The polymorphism ABCB1-rs1045642 was associated with adverse drug reactions under the recessive model (P = 0.017); allele C was associated with no adverse drug reactions, while allele T was associated with adverse drug reactions. The polymorphisms SLC15A1-rs2297322, ABCB1-rs2032582, and CES1-rs2307243 were not consistent with Hardy–Weinberg equilibrium, and no other associations were found for the remaining polymorphisms. In conclusion, the polymorphism rs1045642 in the transporter encoded by the ABCB1 gene is a potential predictive biomarker of ADRs in oseltamivir treatment.

Interaction of Cucurbituril Molecular Containers with the Aerolysin Nanopore for Molecular Recognition

Cucurbit[n]urils (CBn) are molecular containers which form complexes with various guest molecules, such as amino acids and peptides. Here we report the ionic current block and reversible binding of different CBn of sizes n=5, n=6, n=7 and n=8 with the aerolysin (Ael) protein nanopore. CBn molecular containers enter the Ael nanopore exclusively from the extracellular cap domain and under a trans negative voltage. We attempt to relate differential effects of the electrolytes KCl, NaCl, CsCl and LiCl and of the ionic strength on the voltage-dependence of frequency and dwell-time. Notably, the current blockade frequency as well as the current blockade duration increase with increasing the magnitude of applied voltage. Our results suggest that CBn behave as positively charged molecular containers and trans negative voltage promotes their blocking the Ael pore lumen for up to several seconds. Our approach could be used for rapid and real-time probing of the abilities of different CBn to form host-guest complexes with various guest molecules.

Real-Time Estimation of Vehicle Counts on Signalized Intersection Approaches Using Probe Vehicle Data

This paper presents a novel method for estimating the number of vehicles traveling along signalized approaches using probe vehicle data only. The proposed method uses the Kalman Filtering technique to produce reliable vehicle count estimates using real-time probe vehicle estimates of the expected travel times. The proposed method introduces a novel variable estimation interval that allows for higher estimation precision, as the updating time interval always contains a fixed number of probe vehicles. The proposed method is evaluated using empirical and simulated data, the former of which were collected along a signalized roadway in downtown Blacksburg, VA. Results indicate that vehicle count estimates produced by the proposed method are accurate. The paper also examines the model's accuracy when installing a single stationary sensor (e.g., loop detector), producing slight improvements especially when the probe vehicle market penetration rate is low. Finally, the paper investigates the sensitivity of the estimation model to traffic demand levels, showing that the model works better at higher demand levels given that more probe vehicles exist for the same market penetration rate.