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Journal of Ophthalmic & Vision Research 2023Animal models are necessary in understanding the pathogenesis of endophthalmitis and are also necessary to assist the development of new therapeutics for this...
PURPOSE
Animal models are necessary in understanding the pathogenesis of endophthalmitis and are also necessary to assist the development of new therapeutics for this sight-threatening ocular inflammation. Hamilton syringes are usually preferred to inject pathogens when performing experiments on test subjects, however, this method has technical and financial disadvantages. In this study, we report the findings and assess the related benefits of applying a novel low-cost intravitreal injection technique to initiate endophthalmitis in a mouse model while using the Eppendorf tip and a 26G needle.
METHODS
The 18-hr culture of clinical isolates of bacteria ( and ) and fungus ( and ) were resuspended to a final concentration of 10,000 colony forming units (CFU)/1 µL which were separately injected intravitreally into C57BL/6 mice (6-8 weeks) using a 0.1-2.5µL pipette attached to the modified Eppendorf tip with a 26G needle. The contralateral eye served as vehicle/uninjected control. Disease progression was determined by assessing the corneal haze, opacity, bacterial burden, and retinal histology of the eyes used in the model. Following euthanization, bacteria-infected mice were enucleated after 24 hr of the initial injection, and fungus-infected mice after 72 hr.
RESULTS
Of the 50 mice injected, the modified technique was successful in 48 mice. Two mice were excluded due to cataract formed by accidental injury to the lens. The experimental endophthalmitis mice model successfully mimicked the natural clinical course. Clinical assessment and histopathology confirmed the influx of inflammatory cells into the posterior segment of the eye along with dissolution of retinal architecture.
CONCLUSION
Our novel method of injection using a modified Eppendorf tip and 26G needle yielded a cost-effective mouse model of clinical endophthalmitis, resulting in reproducible infection for understanding various aspects of its pathobiology.
PubMed: 37600911
DOI: 10.18502/jovr.v18i3.13775 -
Veterinary World Jan 2024The production of lignocellulosic biomass waste in the agricultural sector of Indonesia is quite high annually. Utilization of lignocellulosic biomass waste through...
BACKGROUND AND AIM
The production of lignocellulosic biomass waste in the agricultural sector of Indonesia is quite high annually. Utilization of lignocellulosic biomass waste through fermentation technology can be used as feed and biofuel. Fermentation technology requires the involvement of micro-organisms such as bacteria (lactic acid bacteria or LAB). LABs can be isolated from various sources, such as duck excreta. However, there have not been many reports of LAB from duck excreta. The present study aimed to characterize LAB enzymes isolated from duck excreta and obtain LAB enzymes with superior fermentation properties.
MATERIALS AND METHODS
A total of 11 LAB cultures obtained from duck excreta in Yogyakarta, Indonesia, were tested. Enzyme characterization of each LAB was performed using the API ZYM kit (BioMérieux, Marcy-I'Etoile, France). The bacterial cell suspension was dropped onto the API ZYM™ cupule using a pipette and incubated for 4 h at 37°C. After incubation, ZYM A and ZYM B were dripped onto the API ZYM cupule, and color changes were observed for approximately 10 s under a strong light source.
RESULTS
Esterase activity was moderate for all LABs. The activity of α-chymotrypsin, β-glucuronidase, α-fucosidase, and α-mannosidase was not observed in a total of 10 LAB. The phosphohydrolase and amino peptidase enzyme activity of seven LABs was strong. Only six LAB samples showed protease activity. The glycosyl hydrolase (GH) activity was observed in a total of 8 LAB, while the activity of 2 LAB was strong ( subsp. K5 and M4A).
CONCLUSION
A total of 2 LABs have superior properties. subsp. K5 and M4A have a high potential to be used in fermentation. They have the potential for further research, such as their effectiveness in fermentation, lignocellulose hydrolysis, feed additives, molecular characterization to detect specific enzymes, and their specific activities.
PubMed: 38406367
DOI: 10.14202/vetworld.2024.143-149 -
Journal of Synchrotron Radiation Jul 2023A sample environment and manipulation tool is presented for single-particle X-ray experiments in an aqueous environment. The system is based on a single water droplet,...
A sample environment and manipulation tool is presented for single-particle X-ray experiments in an aqueous environment. The system is based on a single water droplet, positioned on a substrate that is structured by a hydrophobic and hydrophilic pattern to stabilize the droplet position. The substrate can support several droplets at a time. Evaporation is prevented by covering the droplet by a thin film of mineral oil. In this windowless fluid which minimizes background signal, single particles can be probed and manipulated by micropipettes, which can easily be inserted and steered in the droplet. Holographic X-ray imaging is shown to be well suited to observe and monitor the pipettes, as well as the droplet surface and the particles. Aspiration and force generation are also enabled based on an application of controlled pressure differences. Experimental challenges are addressed and first results are presented, obtained at two different undulator endstations with nano-focused beams. Finally, the sample environment is discussed in view of future coherent imaging and diffraction experiments with synchrotron radiation and single X-ray free-electron laser pulses.
Topics: X-Rays; Radiography; Lasers; Synchrotrons; Holography; Water; X-Ray Diffraction
PubMed: 37233735
DOI: 10.1107/S1600577523003685 -
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 -
Analytica Chimica Acta Feb 2024In "shotgun" approaches involving high-performance liquid chromatography or capillary zone electrophoresis (CZE), matrix removal prior to sample analysis is considered...
BACKGROUND
In "shotgun" approaches involving high-performance liquid chromatography or capillary zone electrophoresis (CZE), matrix removal prior to sample analysis is considered as an indispensable tool. Despite the fact that CZE offers a high tolerance towards salts, most publications reported on the use of desalting. There seems to be no clear consensus on the utilization of desalting in the CZE-MS community, most probably due to the absence of works addressing the comparison of desalted and non-desalted digests. Our aim was to fill this research gap using protein samples of varying complexity in different sample matrices.
RESULTS
First, standard protein digests were analyzed to build the knowledge on the effect of sample clean-up by solid-phase extraction (SPE) pipette tips and the possible stacking phenomena induced by different sample matrices. Desalting led to a somewhat altered peptide profile, the procedure affected mostly the hydrophilic peptides (although not to a devastating extent). Nevertheless, desalting samples allowed remarkable stacking efficiency owing to their low-conductivity sample background, enabling a so-called field-amplified sample stacking phenomenon. Non-desalted samples also produced a stacking event, the mechanism of which is based on transient-isotachophoresis due to the presence of high-mobility ions in the digestion buffer itself. Adding either extra ammonium ions or acetonitrile into the non-desalted digests enhanced the stacking efficiency. A complex sample (yeast cell lysate) was also analyzed with the optimal conditions, which yielded similar tendencies.
SIGNIFICANCE
Based on these results, we propose that sample clean-up in the bottom-up sample preparation process prior to CZE-MS analysis can be omitted. The preclusion of desalting can even enhance detection sensitivity, separation efficiency or sequence coverage.
Topics: Peptide Mapping; Tandem Mass Spectrometry; Proteomics; Electrophoresis, Capillary; Peptides; Ions
PubMed: 38220294
DOI: 10.1016/j.aca.2023.342162 -
Scientific Reports Jan 2024The patch-clamp technique has revolutionized neurophysiology by allowing to study single neuronal excitability, synaptic connectivity, morphology, and the transcriptomic...
The patch-clamp technique has revolutionized neurophysiology by allowing to study single neuronal excitability, synaptic connectivity, morphology, and the transcriptomic profile. However, the throughput in recordings is limited because of the manual replacement of patch-pipettes after each attempt which are often also unsuccessful. This has been overcome by automated cleaning the tips in detergent solutions, allowing to reuse the pipette for further recordings. Here, we developed a novel method of automated cleaning by sonicating the tips within the bath solution wherein the cells are placed, reducing the risk of contaminating the bath solution or internal solution of the recording pipette by any detergent and avoiding the necessity of a separate chamber for cleaning. We showed that the patch-pipettes can be used consecutively at least ten times and that the cleaning process does not negatively impact neither the brain slices nor other patched neurons. This method, combined with automated patch-clamp, highly improves the throughput for single and especially multiple recordings.
Topics: Ultrasonics; Detergents; Neurons; Neurophysiology; Patch-Clamp Techniques
PubMed: 38238544
DOI: 10.1038/s41598-024-51837-7 -
Journal of Chromatography. A May 2024Birds are excellent bioindicators of environmental pollution, and blood provides information on contaminant exposure, although its analysis is challenging because of the...
Optimization and uncertainty assessment of a gas chromatography coupled to Orbitrap mass spectrometry method to determine organic contaminants in blood: A case study of an endangered seabird.
Birds are excellent bioindicators of environmental pollution, and blood provides information on contaminant exposure, although its analysis is challenging because of the low volumes that can be sampled. The objective of the present study was to optimize and validate a miniaturized and functional extraction and analytical method based on gas chromatography coupled to Orbitrap mass spectrometry (GCOrbitrap-MS) for the trace analysis of contaminants in avian blood. Studied compounds included 25 organochlorine pesticides (OCPs), 6 polychlorinated biphenyls (PCBs), 8 polybrominated diphenyl ethers (PBDEs) and 15 polycyclic aromatic hydrocarbons (PAHs). Four extraction and clean-up conditions were optimized and compared in terms of efficiency, accuracy, and uncertainty assessment. Extraction with hexane:dichloromethane and miniaturized Florisil pipette clean-up was the most adequate considering precision and accuracy, time, and costs, and was thereafter used to analyse 20 blood samples of a pelagic seabird, namely the Bermuda petrel (Pterodroma cahow). This species, endemic to the Northwest Atlantic, is among the most endangered seabirds of the region that in the '60 faced a decrease in the breeding success likely linked to a consistent exposure to dichloro-diphenyl-trichloroethane (DDT). Indeed, p,p'-DDE, the main DDT metabolite, was detected in all samples and ranged bewteen 1.13 and 6.87 ng/g wet weight. Other ubiquitous compounds were PCBs (ranging from 0.13 to 6.76 ng/g ww), hexachlorobenzene, and mirex, while PAHs were sporadically detected at low concentrations, and PBDEs were not present. Overall, the extraction method herein proposed allowed analysing very small blood volumes (∼ 100 µL), thus respecting ethical principles prioritising the application of less-invasive sampling protocols, fundamental when studying threatened avian species.
Topics: Animals; Gas Chromatography-Mass Spectrometry; Pesticides; Birds; Hydrocarbons, Chlorinated; Polychlorinated Biphenyls; Halogenated Diphenyl Ethers; Polycyclic Aromatic Hydrocarbons; Endangered Species; Environmental Monitoring; Environmental Pollutants
PubMed: 38604058
DOI: 10.1016/j.chroma.2024.464870 -
Frontiers in Microbiology 2023Swab pooling may allow for more efficient use of point-of-care assays for SARS-CoV-2 detection in settings where widespread testing is warranted, but the effects of...
INTRODUCTION
Swab pooling may allow for more efficient use of point-of-care assays for SARS-CoV-2 detection in settings where widespread testing is warranted, but the effects of pooling on assay performance are not well described.
METHODS
We tested the Thermo-Fisher Accula rapid point-of-care RT-PCR platform with contrived pooled nasal swab specimens.
RESULTS
We observed a higher limit of detection of 3,750 copies/swab in pooled specimens compared to 2,250 copies/swab in individual specimens. Assay performance appeared worse in a specimen with visible nasal mucous and debris, although performance was improved when using a standard laboratory mechanical pipette compared to the transfer pipette included in the assay kit.
CONCLUSION
Clinicians and public health officials overseeing mass testing efforts must understand limitations and benefits of swab or sample pooling, including reduced assay performance from pooled specimens. We conclude that the Accula RT-PCR platform remains an attractive candidate assay for pooling strategies owing to the superior analytical sensitivity compared to most home use and point-of-care tests despite the inhibitory effects of pooled specimens we characterized.
PubMed: 37608952
DOI: 10.3389/fmicb.2023.1219214 -
Korean Journal of Orthodontics Sep 2023To evaluate the effects of an electric toothbrush with vibrational frequencies of 125 Hz and 150 Hz on the orthodontic tooth movement (OTM) rate and the production of...
OBJECTIVE
To evaluate the effects of an electric toothbrush with vibrational frequencies of 125 Hz and 150 Hz on the orthodontic tooth movement (OTM) rate and the production of prostaglandin E2 (PGE2).
METHODS
Out of thirty patients (aged 18-25 years; 16 females and 14 males), ten patients each formed Group A and B, who used electric toothbrushes with 125 Hz and 150 Hz vibrations, respectively. The remaining ten patients (Group C) served as the control group and did not use electric toothbrushes. The rate of OTM and levels of PGE2 using microcapillary pipettes were calculated before the start of retraction (T0), on the 30th day (T1), on the 60th day (T2), and on the 90th day (T3) from the start of retraction in all the groups.
RESULTS
There was a statistically significant difference in the mean OTM values and PGE2 levels in all three groups at different time intervals, with the maximum difference seen in Group B compared to Group A and least in Group C at T1, T2 and T3.
CONCLUSIONS
The rate of OTM and levels of PGE2 were highest in patients who used an electric toothbrush with 150 Hz mechanical vibration compared to those who used an electric toothbrush with 125 Hz mechanical vibration and least in patients who did not use an electric toothbrush. Mechanical vibration led to an increase in the PGE2 levels and accelerated the OTM.
PubMed: 37746776
DOI: 10.4041/kjod23.076 -
American Journal of Physiology. Cell... Jan 2024Airway smooth muscle (ASM) cells from mouse bronchus express a fast sodium current mediated by Na1.7. We present evidence that this current is regulated by cAMP. ASM...
Airway smooth muscle (ASM) cells from mouse bronchus express a fast sodium current mediated by Na1.7. We present evidence that this current is regulated by cAMP. ASM cells were isolated by enzymatic dispersal and studied using the whole cell patch clamp technique at room temperature. A fast sodium current, , was observed on holding cells under voltage clamp at -100 mV and stepping to -20 mV. This current was reduced in a concentration-dependent manner by denopamine (10 and 30 µM), a β-adrenergic agonist. Forskolin (1 µM), an activator of adenylate cyclase, reduced the current by 35%, but 6-MB-cAMP (300 µM), an activator of protein kinase A (PKA), had no effect. In contrast, 8-pCPT-2-O-Me-cAMP-AM (007-AM, 10 µM), an activator of exchange protein directly activated by cAMP (Epac), reduced the current by 48%. The inhibitory effect of 007-AM was still observed in the presence of dantrolene (10 µM), an inhibitor of ryanodine receptors, and when cytosolic [Ca] was buffered by inclusion of 1,2-bis(-aminophenoxy)ethane-,,,-tetraacetic acid, Sigma (BAPTA) (50 µM) in the pipette solution, suggesting that the inhibition of was not due to Ca-release from intracellular stores. When 007-AM was tested on the current-voltage relationship, it reduced the current at potentials from -30 to 0 mV, but had no effect on the steady-state activation curve. However, the steady-state inactivation , the voltage causing inactivation of 50% of the current, was shifted in the negative direction from -76.6 mV to -89.7 mV. These findings suggest that cAMP regulates in mouse ASM via Epac, but not PKA. β-adrenergic agonists are commonly used in inhalers to treat asthma and chronic obstructive pulmonary disease. These work by causing bronchodilation and reducing inflammation. The present study provides evidence that these drugs have an additional action, namely, to reduce sodium influx into airway smooth muscle cells via fast voltage-dependent channels. This may have the dual effect of promoting bronchodilation and reducing remodeling of the airways, which has a detrimental effect in these diseases.
Topics: Mice; Animals; Sodium; Cyclic AMP; Guanine Nucleotide Exchange Factors; Myocytes, Smooth Muscle; Adrenergic beta-Agonists
PubMed: 37955124
DOI: 10.1152/ajpcell.00417.2023