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Methods in Molecular Biology (Clifton,... 2022Synaptic vesicles (SVs) store neurotransmitters and undergo a fine-tuned regulatory and dynamic cycle of exo- and endocytosis, which is essential for neurotransmission...
Synaptic vesicles (SVs) store neurotransmitters and undergo a fine-tuned regulatory and dynamic cycle of exo- and endocytosis, which is essential for neurotransmission at chemical synapses. The development of protocols for isolating SVs from biological extracts was a fundamental accomplishment since it allowed for characterizing the molecular properties of SVs using biochemical methods. In this chapter, we describe a modified procedure for isolating SVs from a few g of rodent brain and that can be completed within ~12 h. The protocol involves the preparation of isolated nerve terminals from which SVs are released by osmotic shock and then enriched via various centrifugation steps, followed by size exclusion chromatography as final purification step. The final vesicle fraction is 22-fold enriched in SVs over the starting material, and the final yield of SVs obtained using this protocol is approximately 20 μg of protein per gram of mouse brain. The degree of contamination by other organelles and particles monitored by morphology and immunolabeling compares well with that of the classical protocols.
Topics: Animals; Brain; Mammals; Mice; Neurotransmitter Agents; Synapses; Synaptic Transmission; Synaptic Vesicles
PubMed: 35099797
DOI: 10.1007/978-1-0716-1916-2_11 -
Veterinary Ophthalmology Apr 2023To assess in vitro antibacterial efficacy of three cross-linking (XL) protocols on bacteria associated with canine ulcerative keratitis.
OBJECTIVE
To assess in vitro antibacterial efficacy of three cross-linking (XL) protocols on bacteria associated with canine ulcerative keratitis.
METHODS
Three XL protocols: UVA 3 mW/cm for 60 min, UVA 3 mW/cm for 30 min, and UVA 30 mW/cm for 3 min with and without application of riboflavin and a riboflavin-only protocol were performed in vitro on the four most common bacterial genera isolated from cases of canine ulcerative keratitis treated at Dick White Referrals, UK. Zones of bacterial growth inhibition (GIZ) associated with treatment were measured and compared.
RESULTS
The four most common isolates were Pseudomonas aeruginosa (PA) (48/140, 34.3%), Streptococcus spp. (32/140, 22.9%), Staphylococcus spp. (24/140, 17.1%) and Escherichia coli (EC) (11/140, 7.9%). PA, EC, Streptococcus canis (SC), and Staphylococcus pseudintermedius (SP), isolated from canine corneas, were selected for testing. EC and SC demonstrated growth inhibition following all UVA/riboflavin protocols. PA and SP only displayed growth inhibition following the 60 min UVA/riboflavin protocol. GIZ areas for 60 min UVA/riboflavin protocols were significantly greater than 30 and 3 min UVA/riboflavin protocols (p < .01) and there was no significant difference between 30 and 3 min UVA/riboflavin protocols. In respect to GIZ areas, EC was significantly more susceptible to XL than SP (p = <.01).
CONCLUSIONS
All UVA/riboflavin XL protocols caused growth inhibition of EC and SC in vitro. PA and SP did not show clear growth inhibition in vitro following exposure to XL protocol settings of UVA 3 mW/cm for 30 min and UVA 30 mW/cm for 3 min.
Topics: Animals; Dogs; Photosensitizing Agents; Corneal Ulcer; Ultraviolet Rays; Cross-Linking Reagents; Cornea; Riboflavin; Bacteria; Corneal Stroma; Dog Diseases
PubMed: 35713165
DOI: 10.1111/vop.13006 -
PeerJ. Computer Science 2023The aim of this article is to identify a range of changes and challenges that present-day technologies often present to contemporary societies, particularly in the...
The aim of this article is to identify a range of changes and challenges that present-day technologies often present to contemporary societies, particularly in the context of smart city logistics, especially during crises. For example, the long-term consequences of the COVID-19 pandemic, such as life losses, economic damages, and privacy and security violations, demonstrate the extent to which the existing designs and deployments of technological means are inadequate. The article proposes a privacy-preserving, decentralized, secure protocol to safeguard individual boundaries and supply governments and public health organizations with cost-effective information, particularly regarding vaccination. The contribution of this article is threefold: (i) conducting a systematic review of most of the privacy-preserving apps and their protocols created during pandemics, and we found that most apps pose security and privacy violations. (ii) Proposing an agent-based, decentralized private set intersection (PSI) protocol for securely sharing individual digital personal and health passport information. The proposed scheme is called secure mobile digital passport agent (SMDPA). (iii) Providing a simulation measurement of the proposed protocol to assess performance. The performance result proves that SMDPA is a practical solution and better than the proposed active data bundles using secure multi-party computation (ADB-SMC), as the average CPU load for SMDPA is approximately 775 milliseconds (ms) compared to about 900 ms for ADB-SMC.
PubMed: 37547404
DOI: 10.7717/peerj-cs.1458 -
Nature Protocols Aug 2021The minimal inhibitory concentration (MIC) assay uses agar or broth dilution methods to measure, under defined test conditions, the lowest effective concentration of an... (Review)
Review
The minimal inhibitory concentration (MIC) assay uses agar or broth dilution methods to measure, under defined test conditions, the lowest effective concentration of an antimicrobial agent that inhibits visible growth of a bacterium of interest. This assay is used to test the susceptibilities of bacterial isolates and of novel antimicrobial drugs, and is typically done in nutrient-rich laboratory media that have little relevance to in vivo conditions. As an extension to our original protocol on MIC assays (also published in Nature Protocols), here we describe the application of the MIC broth microdilution assay to test antimicrobial susceptibility in conditions that are more physiologically relevant to infections observed in the clinic. Specifically, we describe a platform that can be applied to the preparation of medium that mimics lung and wound exudate or blood conditions for the growth and susceptibility testing of bacteria, including ESKAPE pathogens. This protocol can also be applied to most physiologically relevant liquid medium and aerobic pathogens, and takes 3-4 d to complete.
Topics: Animals; Anti-Bacterial Agents; Bacteria; Culture Media; Drug Resistance, Bacterial; Humans; Microbial Sensitivity Tests; Spectrophotometry; Time Factors
PubMed: 34215865
DOI: 10.1038/s41596-021-00572-8 -
Hematology (Amsterdam, Netherlands) Dec 2023Anthracyclines and cytarabine have comprised standard induction therapy for acute myeloid leukemia (AML) for decades. Low overall survival of AML is due to non-remission...
OBJECTIVE
Anthracyclines and cytarabine have comprised standard induction therapy for acute myeloid leukemia (AML) for decades. Low overall survival of AML is due to non-remission or relapse after remission. Hypomethylating agent (HMA) decitabine combined with low-dose chemotherapy or other targeted agents has shown promising effect for AML in clinical trials, especially in (8;21) acute myeloid leukemia. We previously investigated histone deacetylase inhibitor (HDACi) chidamide could regulate Wnt/β-catenin signaling pathway in leukemia cell lines.
METHODS
Adult patients with or relapsed/refractory AML who were treated with chidamide and decitabine in combination with chemotherapy (chidamide group, = 23) or only decitabine combination with chemotherapy (decitabine group, = 17) were analyzed.
RESULTS
Chidamide group represented higher complete response rate (82.6% and 52.9%, 0.0430, decitabine group), progression-free survival and overall survival rates (= 0.0088 and = 0.0139, respectively), especially for patients with AML. Hematological toxicity and infections were the most common adverse events (AEs) in both groups, and they were manageable by supportive treatments.
CONCLUSIONS
This HDACi- and HMA-based protocol is an effective and tolerable therapy for patients with AML. The comprehensive mechanism and effects of chidamide in combination with decitabine are worth to be further explored in AML.
Topics: Adult; Humans; Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Cytarabine; Decitabine; Histone Deacetylase Inhibitors; Leukemia, Myeloid, Acute; Retrospective Studies; Antimetabolites, Antineoplastic
PubMed: 37278601
DOI: 10.1080/16078454.2023.2219930 -
Compendium of Continuing Education in... 2023The clinical quality and longevity of increasingly popular resin-bonded CAD/CAM ceramic restorations depends greatly on the strength and durability of the resin-ceramic...
The clinical quality and longevity of increasingly popular resin-bonded CAD/CAM ceramic restorations depends greatly on the strength and durability of the resin-ceramic bond. With a diverse array of ceramic materials available, the choice of ceramic bonding protocol is highly influenced by the specific ceramic's type and composition. Typically, this protocol encompasses a surface pretreatment step followed by the application of a priming agent. This article provides a comprehensive update on current resin-bonding protocols for the most commonly used CAD/CAM ceramics.
Topics: Dental Bonding; Resin Cements; Surface Properties; Materials Testing; Ceramics; Computer-Aided Design; Shear Strength; Dental Porcelain
PubMed: 38133889
DOI: No ID Found -
Bratislavske Lekarske Listy 2020Bacillus anthracis is a causative agent of zoonotic anthrax disease. In the last years, significant progress in therapy and diagnosis of anthrax was made. Concurrently,... (Review)
Review
AIM
Bacillus anthracis is a causative agent of zoonotic anthrax disease. In the last years, significant progress in therapy and diagnosis of anthrax was made. Concurrently, knowledge about anthrax progression, molecular pathology and release of anthrax toxin during the disease has improved. This review covers the recent progress in this field.
METHODS
In this review, specifications of B. anthracis, anthrax disease, medical and biomedical countermeasures and diagnostic tools were surveyed. The actual literature was summarized and relevance of the microorganism as a biological warfare agent and the ways how to reduce its impact including therapeutic protocols were written and discussed.
RESULTS
Currently, the microorganism is considered one of the top biological warfare agents due to lethality, long term stability of spores, easy dissemination and production. The recent research is focused on countermeasures suitable for reduction of consequences by a misuse of the microorganism in form of biological weapon (Tab. 3, Fig. 1, Ref. 101). Text in PDF www.elis.sk.
Topics: Anthrax; Bacillus anthracis; Biological Warfare Agents; Humans
PubMed: 32115973
DOI: 10.4149/BLL_2020_026 -
Formal Methods in System Design 2021Population protocols are a well established model of computation by anonymous, identical finite-state agents. A protocol is well-specified if from every initial...
Population protocols are a well established model of computation by anonymous, identical finite-state agents. A protocol is well-specified if from every initial configuration, all fair executions of the protocol reach a common consensus. The central verification question for population protocols is the : deciding if a given protocol is well-specified. Esparza et al. have recently shown that this problem is decidable, but with very high complexity: it is at least as hard as the Petri net reachability problem, which is TOWER-hard, and for which only algorithms of non-primitive recursive complexity are currently known. In this paper we introduce the class of well-specified strongly-silent protocols and we prove that it is suitable for automatic verification. More precisely, we show that has the same computational power as general well-specified protocols, and captures standard protocols from the literature. Moreover, we show that the membership and correctness problems for reduce to solving boolean combinations of linear constraints over . This allowed us to develop the first software able to automatically prove correctness for of the infinitely many possible inputs.
PubMed: 34866798
DOI: 10.1007/s10703-021-00367-3 -
Methods and Applications in Fluorescence Mar 2021Optical tissue clearing refers to physico-chemical treatments which make thick biological samples transparent by removal of refractive index gradients and light... (Review)
Review
Optical tissue clearing refers to physico-chemical treatments which make thick biological samples transparent by removal of refractive index gradients and light absorbing substances. Although tissue clearing was first reported in 1914, it was not widely used in light microscopy until 21th century, because instrumentation of that time did not permit to acquire and handle images of thick (mm to cm) samples as whole. Rapid progress in optical instrumentation, computers and software over the last decades made micrograph acquisition of centimeter-thick samples feasible. This boosted tissue clearing use and development. Numerous diverse protocols have been developed. They use organic solvents or water-miscible substances, such as detergents and chaotropic agents; some protocols require application of electric field or perfusion with special devices. There is no 'best-for-all' tissue clearing method. Depending on the case, one or another protocol is more suitable. Most of protocols require days or even weeks to complete, thus choosing an unsuitable protocol may cause an important waste of time. Several inter-dependent parameters should be taken into account to choose a tissue clearing protocol, such as: (1) required image quality (resolution, contrast, signal to noise ratio etc), (2) nature and size of the sample, (3) type of labels, (4) characteristics of the available instrumentation, (5) budget, (6) time budget, and (7) feasibility. Present review focusses on the practical aspects of various tissue clearing techniques. It is aimed to help non-experts to choose tissue clearing techniques which are optimal for their particular cases.
Topics: Animals; Histocytological Preparation Techniques; Humans; Microscopy
PubMed: 33592593
DOI: 10.1088/2050-6120/abe6fb -
Proteomes Mar 2023Integrative top-down proteomics is an analytical approach that fully addresses the breadth and complexity needed for effective and routine assessment of proteomes....
Integrative top-down proteomics is an analytical approach that fully addresses the breadth and complexity needed for effective and routine assessment of proteomes. Nonetheless, any such assessments also require a rigorous review of methodology to ensure the deepest possible quantitative proteome analyses. Here, we establish an optimized general protocol for proteome extracts to improve the reduction of proteoforms and, thus, resolution in 2DE. Dithiothreitol (DTT), tributylphosphine (TBP), and 2-hydroxyethyldisulfide (HED), combined and alone, were tested in one-dimensional SDS-PAGE (1DE), prior to implementation into a full 2DE protocol. Prior to sample rehydration, reduction with 100 mM DTT + 5 mM TBP yielded increased spot counts, total signal, and spot circularity (i.e., decreased streaking) compared to other conditions and reduction protocols reported in the literature. The data indicate that many widely implemented reduction protocols are significantly 'under-powered' in terms of proteoform reduction and thus, limit the quality and depth of routine top-down proteomic analyses.
PubMed: 36976889
DOI: 10.3390/proteomes11010010