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Brain Stimulation 2023Focused ultrasound (FUS) is a medical technology that non-invasively stimulates the brain and has been applied in thermal ablation, blood-brain barrier (BBB) opening,...
BACKGROUND
Focused ultrasound (FUS) is a medical technology that non-invasively stimulates the brain and has been applied in thermal ablation, blood-brain barrier (BBB) opening, and neuromodulation. In recent years, numerous experiences and indications for the use of FUS in clinical and preclinical studies have rapidly expanded. Focused ultrasound-mediated BBB opening induces cognitive enhancement and neurogenesis; however, the underlying mechanisms have not been elucidated.
METHODS
Here, we investigate the effects of FUS-mediated BBB opening on hippocampal long-term potentiation (LTP) and cognitive function in a 5xFAD mouse model of Alzheimer's disease (AD). We applied FUS with microbubble to the hippocampus and LTP was measured 6 weeks after BBB opening using FUS. Field recordings were made with a concentric bipolar electrode positioned in the CA1 region using an extracellular glass pipette filled with artificial cerebrospinal fluid. Morris water maze and Y-maze was performed to test cognitive function.
RESULTS
Our results demonstrated that FUS-mediated BBB opening has a significant impact on increasing LTP at Schaffer collateral - CA1 synapses and rescues cognitive dysfunction and working memory. These effects persisted for up to 7 weeks post-treatment. Also, FUS-mediated BBB opening in the hippocampus increased PKA phosphorylation.
CONCLUSION
Therefore, it could be a promising treatment for neurodegenerative diseases as it remarkably increases LTP, thereby improving working memory.
Topics: Mice; Animals; Alzheimer Disease; Brain; Hippocampus; Neuronal Plasticity; Memory, Short-Term
PubMed: 37211337
DOI: 10.1016/j.brs.2023.05.014 -
Scientific Reports May 2023Accurate interpretation of third harmonic generation (THG) microscopy images in terms of sample optical properties and microstructure is generally hampered by the...
Accurate interpretation of third harmonic generation (THG) microscopy images in terms of sample optical properties and microstructure is generally hampered by the presence of excitation field distortions resulting from sample heterogeneity. Numerical methods that account for these artifacts need to be established. In this work, we experimentally and numerically analyze the THG contrast obtained from stretched hollow glass pipettes embedded in different liquids. We also characterize the nonlinear optical properties of 2,2[Formula: see text]-thiodiethanol (TDE), a water-soluble index-matching medium. We find that index discontinuity not only changes the level and modulation amplitude of polarization-resolved THG signals, but can even change the polarization direction producing maximum THG near interfaces. We then show that a finite-difference time-domain (FDTD) modeling strategy can accurately account for contrast observed in optically heterogeneous samples, whereas reference Fourier-based numerical approaches are accurate only in the absence of index mismatch. This work opens perspectives for interpreting THG microscopy images of tubular objects and other geometries.
PubMed: 37188736
DOI: 10.1038/s41598-023-34528-7 -
Microbiology Spectrum Jun 2023Hepatitis A diagnosis relies on serology and occasionally on hepatitis A virus (HAV) RNA detection. For timely diagnosis and the avoidance of drawing additional blood,...
Sporadic Hepatitis A Virus PCR False-Positive Results Observed during Reflex Testing of Serum Samples Previously Tested for Anti-HAV Antibodies and Caused by Contamination with HAV RNA Present in the Reagents of the Commercial Anti-HAV Immunoassay.
Hepatitis A diagnosis relies on serology and occasionally on hepatitis A virus (HAV) RNA detection. For timely diagnosis and the avoidance of drawing additional blood, molecular testing is often performed as reflex testing by using blood specimens that were initially sent for anti-HAV serology. Reflex molecular testing is preferably performed from different sample aliquots, but, for limited sample quantities, it uses samples that have been preprocessed in an immunoassay analyzer. In 2012, we first observed sporadic HAV RNA-positive cases that were inconsistent with patients' serological profiles and/or medical histories, suggesting that occasional laboratory contamination was causing false-positive PCR results. Multiple external quality assurance (EQA) and laboratory surface contamination checks were performed, questionable specimens were tested with various HAV RNA tests, and follow-up serum/stool samples were collected. All contamination-check samples and samples from healthy individuals tested HAV RNA-negative, and the laboratory successfully passed all EQAs. The HAV RNA-positive results were reproducible with various HAV RNA assays. No patients seroconverted, and their follow-up samples were consistently HAV RNA-negative. Finally, a detailed review of testing protocols revealed a correlation between HAV RNA false positivity and preceding anti-HAV testing with the Cobas-e411 automated immunoassay analyzer. HAV RNA was detected in the Cobas-e411 anti-HAV reagents, with the HAV sequences matching those from the false-positive samples. Preceding anti-HAV testing using two other immunoassay analyzers did not result in subsequent HAV RNA false positivity during reflex testing. The Cobas-e411 pipetting procedure with a single pipette tip collecting samples and anti-HAV reagents contaminated the original sample with the HAV RNA that was present in the immunoassay's reagents, thereby resulting in HAV RNA false positivity during the reflex testing. We present the first report of sporadic HAV PCR false-positive results that have been observed during the reflex testing of serum samples that have previously been tested for anti-HAV antibodies and have been caused by contamination with HAV RNA that is present in the reagents of the commercial anti-HAV immunoassay, with potentially serious clinical consequences. Although HAV RNA was consistently detected in the anti-HAV reagents of all three automated immunoassay analyzers that were in use in our laboratory, only the use of one analyzer and the corresponding commercial anti-HAV immunoassay reagents resulted in contamination that led to false positive HAV RNA results, and this was due to a peculiar pipetting mode of action in which the analyzer uses a single pipette tip to collect both anti-HAV reagents and a sample, which consequently causes the permanent contamination of the original sample with HAV RNA. Manufacturers should strongly consider the occasional need for reflex molecular testing from preprocessed samples and design their analyzers in a way that prevents contamination.
Topics: Humans; Hepatitis A virus; Hepatitis A; Hepatitis A Antibodies; Indicators and Reagents; RNA, Viral; Polymerase Chain Reaction; Immunoassay; Reflex
PubMed: 37162362
DOI: 10.1128/spectrum.00122-23 -
JBRA Assisted Reproduction Sep 2023Endogenous prostaglandins are involved in hemostasis, renal excretion of electrolytes, and implicated in dysmenorrhea. Piroxicam and Nitroglycerin are common drugs used...
OBJECTIVE
Endogenous prostaglandins are involved in hemostasis, renal excretion of electrolytes, and implicated in dysmenorrhea. Piroxicam and Nitroglycerin are common drugs used in treating dysmenorrhea by inhibiting the cyclooxygenase pathway involved in prostaglandin production. However, studies comparing the effects of these drugs on prostaglandin-modulated hemostasis and renal function are lacking.
METHODS
Fifteen female rats (120-160g) were divided into 3 groups (20 per group), namely Control (distilled water, 0.3 mL), Piroxicam treated (3mg/kg) and Nitroglycerin treated (1 mg/kg). Di-estrous phase was confirmed in animals in each group using the Pipette smear method. Treatment was administered for 4 days covering the estrous cycle. Bleeding and clotting time were assessed and blood concentrations of sodium, potassium, urea and platelet counts were evaluated in all phases. Data were analyzed using one-way ANOVA and Newman-Keuls post-hoc test. Statistical significance was considered at p<0.0.
RESULTS
The nitroglycerin-treated group showed significant increases in blood potassium during di-estrous while the piroxicam-treated group showed significant increases in blood potassium, urea and clotting time with a significant decrease in sodium levels during di-estrous compared to controls. Results obtained in other phases were not significant compared to controls.
CONCLUSIONS
The study showed that Nitroglycerin produces minimum alteration of blood and electrolyte indices compared to piroxicam during di-estrous.
Topics: Humans; Female; Rats; Animals; Piroxicam; Rats, Wistar; Nitroglycerin; Cyclooxygenase Inhibitors; Prostaglandins; Dysmenorrhea; Urea; Sodium; Electrolytes; Potassium
PubMed: 37134015
DOI: 10.5935/1518-0557.20220069 -
Advances in Respiratory Medicine Apr 2023Respiratory diseases such as cystic fibrosis, COPD, and COVID-19 are difficult to treat owing to viscous secretions in the airways that evade mucocilliary clearance....
Respiratory diseases such as cystic fibrosis, COPD, and COVID-19 are difficult to treat owing to viscous secretions in the airways that evade mucocilliary clearance. Earlier studies have shown success with BromAc as a mucolytic agent. Hence, we tested the formulation on two gelatinous airway representative sputa models, to determine whether similar efficacy exist. Sputum lodged in an endotracheal tube was treated to aerosol N-acetylcysteine, bromelain, or their combination (BromAc). After measuring the particle size of aerosolized BromAc, the apparent viscosity was measured using a capillary tube method, whilst the sputum flow was assessed using a 0.5 mL pipette. Further, the concentration of the agents in the sputa after treatment were quantified using chromogenic assays. The interaction index of the different formulations was also determined. Results indicated that the mean particle size of BromAc was suitable for aerosol delivery. Bromelain and N-acetylcysteine affected both the viscosities and pipette flow in the two sputa models. BromAc showed a greater rheological effect on both the sputa models compared to individual agents. Further, a correlation was found between the rheological effects and the concentration of agents in the sputa. The combination index using viscosity measurements showed synergy only with 250 µg/mL bromelain + 20 mg/mL NAC whilst flow speed showed synergy for both combinations of bromelain (125 and 250 µg/mL) with 20 mg/mL NAC. Hence, this study indicates that BromAc may be used as a successful mucolytic for clearing airway congestion caused by thick mucinous immobile secretions.
Topics: Humans; Acetylcysteine; Sputum; Bromelains; COVID-19; Expectorants; Respiration Disorders; Rheology
PubMed: 37102780
DOI: 10.3390/arm91020013 -
Microbiology Spectrum Jun 2023Carryover contamination during amplicon sequencing workflow (AMP-Seq) put the accuracy of the high-throughput detection for pathogens at risk. The purpose of this study...
Carryover contamination during amplicon sequencing workflow (AMP-Seq) put the accuracy of the high-throughput detection for pathogens at risk. The purpose of this study is to develop a carryover contaminations-controlled AMP-Seq (ccAMP-Seq) workflow to enable accurate qualitative and quantitative detection for pathogens. By using the AMP-Seq workflow to detect SARS-CoV-2, Aerosols, reagents and pipettes were identified as potential sources of contaminations and ccAMP-Seq was then developed. ccAMP-Seq used filter tips and physically isolation of experimental steps to avoid cross contamination, synthetic DNA spike-ins to compete with contaminations and quantify SARS-CoV-2, dUTP/uracil DNA glycosylase system to digest the carryover contaminations, and a new data analysis procedure to remove the sequencing reads from contaminations. Compared to AMP-Seq, the contamination level of ccAMP-Seq was at least 22-folds lower and the detection limit was also about an order of magnitude lower-as low as one copy/reaction. By testing the dilution series of SARS-CoV-2 nucleic acid standard, ccAMP-Seq showed 100% sensitivity and specificity. The high sensitivity of ccAMP-Seq was further confirmed by the detection of SARS-CoV-2 from 62 clinical samples. The consistency between qPCR and ccAMP-Seq was 100% for all the 53 qPCR-positive clinical samples. Seven qPCR-negative clinical samples were found to be positive by ccAMP-Seq, which was confirmed by extra qPCR tests on subsequent samples from the same patients. This study presents a carryover contamination-controlled, accurate qualitative and quantitative amplicon sequencing workflow that addresses the critical problem of pathogen detection for infectious diseases. Accuracy, a key indicator of pathogen detection technology, is compromised by carryover contamination in the amplicon sequencing workflow. Taking the detection of SARS-CoV-2 as case, this study presents a new carryover contamination-controlled amplicon sequencing workflow. The new workflow significantly reduces the degree of contamination in the workflow, thereby significantly improving the accuracy and sensitivity of the SARS-CoV-2 detection and empowering the ability of quantitative detection. More importantly, the use of the new workflow is simple and economical. Therefore, the results of this study can be easily applied to other microorganism, which has great significance for improving the detection level of microorganism.
Topics: Humans; SARS-CoV-2; COVID-19; Workflow; Sensitivity and Specificity; High-Throughput Nucleotide Sequencing
PubMed: 37098913
DOI: 10.1128/spectrum.00206-23 -
The Science of the Total Environment Jul 2023A rapid virus concentration method is needed to get high throughput. Reliable results of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection in...
A rapid virus concentration method is needed to get high throughput. Reliable results of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection in wastewater are necessary for applications in wastewater-based epidemiology. In this study, an automated filtration method using a concentrating pipette (CP Select; Innovaprep) was applied to detect SARS-CoV-2 in wastewater samples with several modifications to increase its sensitivity and throughput. The performance of the CP Select method was compared to other concentration methods (polyethylene glycol precipitation and direct capture using silica column) to evaluate its applicability to SARS-CoV-2 detection in wastewater. SARS-CoV-2 RNA was successfully detected in six of eight wastewater samples using the CP Select method, whereas other methods could detect SARS-CoV-2 RNA in all wastewater samples. Enteric viruses, such as noroviruses of genogroups I (NoVs-GI) and II (NoVs-GII) and enteroviruses, were tested, resulting in 100 % NoVs-GII detection using all concentration methods. As for NoVs-GI and enteroviruses, all methods gave comparable number of detected samples in wastewater samples. This study showed that the optimized CP Select method was less sensitive in SARS-CoV-2 detection in wastewater than other methods, whereas all methods were applicable to detect or recover other viruses in wastewater.
Topics: Humans; SARS-CoV-2; Wastewater; COVID-19; RNA, Viral; Viruses; Norovirus; Enterovirus
PubMed: 37068668
DOI: 10.1016/j.scitotenv.2023.163487 -
International Journal of Molecular... Mar 2023Physiological activation by light of the TRP and TRP-like (TRPL) channels requires the activation of phospholipase Cβ (PLC). The hydrolysis of phosphatidylinositol...
Physiological activation by light of the TRP and TRP-like (TRPL) channels requires the activation of phospholipase Cβ (PLC). The hydrolysis of phosphatidylinositol 4,5, bisphosphate (PIP) by PLC is a crucial step in the still-unclear light activation, while the generation of Diacylglycerol (DAG) by PLC seems to be involved. In this study, we re-examined the ability of a DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) to activate the TRPL channels expressed in HEK cells. Unlike previous studies, we added OAG into the cytosol via a patch-clamp pipette and observed robust activation of the expressed TRPL channels. However, TRPL channel activation was much slower than the physiologically activated TRPL by light. Therefore, we used a picosecond-fast optically activated DAG analogue, OptoDArG. Inactive OptoDArG was added into the intracellular solution with the patch-clamp pipette, and it slowly accumulated on the surface membrane of the recorded HEK cell in the dark. A fast application of intense UV light to the recorded cell resulted in a robust and relatively fast TRPL-dependent current that was greatly accelerated by the constitutively active TRPL pore-region mutation. However, this current of the mutant channel was still considerably slower than the native light-induced TRPL current, suggesting that DAG alone is not sufficient for TRPL channel activation under physiological conditions.
Topics: Animals; Diglycerides; Drosophila; Drosophila Proteins; Light; Membranes; Phosphatidylinositols; Transient Receptor Potential Channels
PubMed: 37047261
DOI: 10.3390/ijms24076289 -
Annals of Biomedical Engineering May 2023Left atrial ligation (LAL) of the chick embryonic heart is a model of the hypoplastic left heart syndrome (HLHS) where a purely mechanical intervention without genetic...
Left atrial ligation (LAL) of the chick embryonic heart is a model of the hypoplastic left heart syndrome (HLHS) where a purely mechanical intervention without genetic or pharmacological manipulation is employed to initiate cardiac malformation. It is thus a key model for understanding the biomechanical origins of HLHS. However, its myocardial mechanics and subsequent gene expressions are not well-understood. We performed finite element (FE) modeling and single-cell RNA sequencing to address this. 4D high-frequency ultrasound imaging of chick embryonic hearts at HH25 (ED 4.5) were obtained for both LAL and control. Motion tracking was performed to quantify strains. Image-based FE modeling was conducted, using the direction of the smallest strain eigenvector as the orientations of contractions, the Guccione active tension model and a Fung-type transversely isotropic passive stiffness model that was determined via micro-pipette aspiration. Single-cell RNA sequencing of left ventricle (LV) heart tissues was performed for normal and LAL embryos at HH30 (ED 6.5) and differentially expressed genes (DEG) were identified.After LAL, LV thickness increased by 33%, strains in the myofiber direction increased by 42%, while stresses in the myofiber direction decreased by 50%. These were likely related to the reduction in ventricular preload and underloading of the LV due to LAL. RNA-seq data revealed potentially related DEG in myocytes, including mechano-sensing genes (Cadherins, NOTCH1, etc.), myosin contractility genes (MLCK, MLCP, etc.), calcium signaling genes (PI3K, PMCA, etc.), and genes related to fibrosis and fibroelastosis (TGF-β, BMP, etc.). We elucidated the changes to the myocardial biomechanics brought by LAL and the corresponding changes to myocyte gene expressions. These data may be useful in identifying the mechanobiological pathways of HLHS.
Topics: Humans; Hypoplastic Left Heart Syndrome; Biomechanical Phenomena; Atrial Fibrillation; Myocardium; Heart Atria; Heart Ventricles
PubMed: 37032398
DOI: 10.1007/s10439-023-03187-0 -
Analytical and Bioanalytical Chemistry Jul 2023In this work, we present an in situ droplet-based derivatization method for fast tissue lipid profiling at multiple isomer levels. On-tissue derivatization for isomer...
In this work, we present an in situ droplet-based derivatization method for fast tissue lipid profiling at multiple isomer levels. On-tissue derivatization for isomer characterization was achieved in a droplet delivered by the TriVersa NanoMate LESA pipette. The derivatized lipids were then extracted and analyzed by the automated chip-based liquid extraction surface analysis (LESA) mass spectrometry (MS) followed by tandem MS to produce diagnostic fragment ions to reveal the lipid isomer structures. Three reactions, i.e., mCPBA epoxidation, photocycloaddition catalyzed by the photocatalyst Ir[dF(CF)ppy](dtbbpy)PF, and Mn(II) lipid adduction, were applied using the droplet-based derivatization to provide lipid characterization at carbon-carbon double-bond positional isomer and sn-positional isomer levels. Relative quantitation of both types of lipid isomers was also achieved based on diagnostic ion intensities. This method provides the flexibility of performing multiple derivatizations at different spots in the same functional region of an organ for orthogonal lipid isomer analysis using a single tissue slide. Lipid isomers were profiled in the cortex, cerebellum, thalamus, hippocampus, and midbrain of the mouse brain and 24 double-bond positional isomers and 16 sn-positional isomers showed various distributions in those regions. This droplet-based derivatization of tissue lipids allows fast profiling of multi-level isomer identification and quantitation and has great potential in tissue lipid studies requiring rapid sample-to-result turnovers.
Topics: Mice; Animals; Tandem Mass Spectrometry; Isomerism; Lipids
PubMed: 37017722
DOI: 10.1007/s00216-023-04653-3