Standard Preparation Procedure Research Articles

Regulatory Science Perspective on the Analysis of Microplastics and Nanoplastics in Human Food.

Microplastics are increasingly reported, not only in the environment but also in a wide range of food commodities. While studies on microplastics in food abound, the current state of science is limited in its application to regulatory risk assessment by a continued lack of standardized definitions, reference materials, sample collection and preparation procedures, fit-for purpose analytical methods for real-world and environmentally relevant plastic mixtures, and appropriate quality controls. This is particularly the case for nanoplastics. These methodological challenges hinder robust, quantitative exposure assessments of microplastic and nanoplastic mixtures from food consumption. Furthermore, limited toxicological studies on whether microplastics and nanoplastics adversely impact human health are also impeded by methodology challenges. Food safety regulatory agencies must consider both the exposure and the risk of contaminants of emerging concern to ascertain potential harm. Foundational to this effort is access to and application of analytical methods with the capability to quantify and characterize micro- and nanoscale sized polymers in complex food matrices. However, the early stages of method development and application of early stage methods to study the distribution and potential health effects of microplastics and nanoplastics in food have largely been done without consideration of the stringent requirements of methods to inform regulatory activities. We provide regulatory science perspectives on the state of knowledge regarding the occurrence of microplastics and nanoplastics in food and present our general approach for developing, validating, and implementing analytical methods for regulatory purposes.

Standardization of the preparation of a herbo-mineral formulation Shwasakasari Rasa

Shwasakasari Rasa is mentioned in Rasa Yoga Sagar,[1] (part 2) as a remedy of Shwasa & Kaasa Roga. This type of Rasa Aushadhi are more effective against bacteria. These drugs have a multi dimension effect like Broad-spectrum antibiotics. In this study an endeavor has been made to determine standard procedure for preparation of the research formulation i.e. Shwasakasari Rasa. SOPs (Standard Operating Procedure) for pharmaceuticals are associated with quality assurance, internal control, production maintenance, utility and human resources or also defined as established or prescribed methods to be strictly followed for the preparation of particular formulations by a selected method. In this section of the research study all the processes adapted for the preparation of Shwasakasari Rasa are well elaborated with explicit detail of quantity, duration, heating pattern, temperature, material & methods together with timely observed and recorded values. Therefore, Shwasakasari Rasa was prepared in the department & analyse all contents present in Rasa Aushadhi for the establishment of drug for the benefit of society and mankind.

Transcutaneous Osseointegrated Prosthesis Systems (TOPS) for Rehabilitation After Lower Limb Loss: Surgical Pearls.

Transcutaneous Osseointegrated Prosthesis Systems (TOPS) for Rehabilitation After Lower Limb Loss: Surgical Pearls.

Ultrasensitive determination of selenium in food samples and its speciation in water and beverages using thiosemicarbazide-incorporated graphene and total-reflection X-ray fluorescence spectrometry

The paper presents a new analytical procedure for the determination and speciation of trace and ultratrace selenium in water, beverages, seafood, milk, and vegetables. The developed method is based on the dispersive micro-solid phase extraction with the use of new thiosemicarbazide-incorporated graphene as a solid sorbent, in combination of the total-reflection X-ray fluorescence spectrometry (TXRF). As a result, we have created an auspicious analytical tool for fast and sensitive analysis of samples with a complex matrix. Regardless of the specimen type, the method is characterized by a very low detection limit of 1.7 pg mL−1 and high precision. The developed strategy allowed us to solve common problems associated with selenium loss during the sample preparation for the TXRF measurement and also improve its performance toward the analysis of beverages and high saline/solid samples, which may even be impossible to perform using standard sample preparation procedures for a TXRF measurement.

Proteomics-Driven Biomarkers in Pancreatic Cancer.

Pancreatic cancer is a devastating disease that has a grim prognosis, highlighting the need for improved screening, diagnosis, and treatment strategies. Currently, the sole biomarker for pancreatic ductal adenocarcinoma (PDAC) authorized by the U.S. Food and Drug Administration is CA 19-9, which proves to be the most beneficial in tracking treatment response rather than in early detection. In recent years, proteomics has emerged as a powerful tool for advancing our understanding of pancreatic cancer biology and identifying potential biomarkers and therapeutic targets. This review aims to offer a comprehensive survey of proteomics' current status in pancreatic cancer research, specifically accentuating its applications and its potential to drastically enhance screening, diagnosis, and treatment response. With respect to screening and diagnostic precision, proteomics carries the capacity to augment the sensitivity and specificity of extant screening and diagnostic methodologies. Nonetheless, more research is imperative for validating potential biomarkers and establishing standard procedures for sample preparation and data analysis. Furthermore, proteomics presents opportunities for unveiling new biomarkers and therapeutic targets, as well as fostering the development of personalized treatment strategies based on protein expression patterns associated with treatment response. In conclusion, proteomics holds great promise for advancing our understanding of pancreatic cancer biology and improving patient outcomes. It is essential to maintain momentum in investment and innovation in this arena to unearth more groundbreaking discoveries and transmute them into practical diagnostic and therapeutic strategies in the clinical context.

Study of microstructural, chemical and microhardness characterization of stainless steels

Today it is almost impossible to find any area of human activity in which metal would not be present and would be closely related to development. Therefore, it is necessary to know its properties and what kind of media will be used in order to select the best type of material to use. This study examines some of the various austenitic stainless steels in terms of composition, microstructure, and hardness. For these analyses, it was necessary to follow a series of standard sample preparation procedures that directly affect the exact result, microstructure, and hardness. The percentage of chromium varied from 17 to 18%, which gives the material good passivity. Molybdenum in grades 304 and 304L is presented as a residual content, and in grades 316 and 316L - as an alloying element. Among all the studied materials, AISI 304 showed the highest hardness. The relationship between the alloy composition, microstructure and hardness, and the specific properties of each steel is discussed.

In-ovo echocardiography for application in cardiovascular research

Preclinical cardiovascular research relies heavily on non-invasive in-vivo echocardiography in mice and rats to assess cardiac function and morphology, since the complex interaction of heart, circulation, and peripheral organs are challenging to mimic ex-vivo. While n-numbers of annually used laboratory animals worldwide approach 200 million, increasing efforts are made by basic scientists aiming to reduce animal numbers in cardiovascular research according to the 3R’s principle. The chicken egg is well-established as a physiological correlate and model for angiogenesis research but has barely been used to assess cardiac (patho-) physiology. Here, we tested whether the established in-ovo system of incubated chicken eggs interfaced with commercially available small animal echocardiography would be a suitable alternative test system in experimental cardiology. To this end, we defined a workflow to assess cardiac function in 8–13-day-old chicken embryos using a commercially available high resolution ultrasound system for small animals (Vevo 3100, Fujifilm Visualsonics Inc.) equipped with a high frequency probe (MX700; centre transmit: 50 MHz). We provide detailed standard operating procedures for sample preparation, image acquisition, data analysis, reference values for left and right ventricular function and dimensions, and inter-observer variabilities. Finally, we challenged incubated chicken eggs with two interventions well-known to affect cardiac physiology—metoprolol treatment and hypoxic exposure—to demonstrate the sensitivity of in-ovo echocardiography. In conclusion, in-ovo echocardiography is a feasible alternative tool for basic cardiovascular research, which can easily be implemented into the small animal research environment using existing infrastructure to replace mice and rat experiments, and thus, reduce use of laboratory animals according to the 3R principle.

Mechanical characterisation of new Sand-Hemihydrate rock-analogue material: Implications for modelling of brittle crust processes

The analogue materials play a critical role in dynamically scaled experiments, defining the processes that can be simulated and the structures observable in the model. The dynamic scaling enables the direct comparison between the model and its natural counterpart. To obtain such a model the physical and mechanical properties of the analogue material must be scaled with respect to the rock prototype. A large variety of materials have been applied in analogue modelling studies to address the physical and mechanical requirements for the simulation of (i) upper crust, (ii) middle crust and (iii) lower crust and mantle processes. Nevertheless, the development of new model materials represents a continuous improvement of the analogue modelling techniques.We investigated the mechanical and physical properties of a new Granular Rock-Analogue Material (GRAM) (introduced in Massaro et al. (2022)) and its component materials. GRAM is an ultra-weak artificial sandstone composed of quartz sand cemented with gypsum, capable to deform by tensile and shear failure under variable stress conditions. GRAM aggregates in different mixing ratios (from 1% to 4% in weight of hemihydrate powder) were systematically tested with ring-shear tests and uniaxial compression tests. The relationships between the hemihydrate content and the mechanical properties of GRAM were examined. Additionally, the sample preparation procedure of GRAM was investigated, evaluating the impact of the residual water content on the mechanical properties of GRAM aggregates and defining a standard preparation procedure. Finally, GRAM was compared to natural rocks and to other granular materials applied in analogue modelling studies, in terms of physical and mechanical properties, application to physical modelling and dynamic scaling. It was underlined how the application of GRAM aggregates in dynamically scaled experiments can enhance the comprehension of the fault and fracture processes occurring at the scale of the damage zone.

Optimization of sample preparation conditions in the determination of total arsenic in fish and seafoods by atomic absorption spectrometry with electrothermal atomization

The conditions for sample preparation and determination of total arsenic in fish and seafoods by atomic absorption spectroscopy with electrothermal atomization have been optimized. When using a standard sample preparation procedure (GOST R 53100), a complete decomposition of the organic arsenic compound, arsenobetaine, appeared impossible which leads to underestimated results. A novel sample preparation procedure for the determination of total arsenic, including the addition of concentrated sulfuric acid for complete decomposition of the organic forms of arsenic to inorganic ones is proposed. During the atomization of the sample, an additional stage of drying was introduced with smooth heating from 200 to 250°C, which contributed to a complete removal of sulfuric acid residues from the injected sample volume. The correctness of the proposed methodology was verified using the ICP-MS method and in the analysis of reference samples of fish and seafoods.

Leaching of dissolved organic carbon from commercial reverse osmosis membranes exacerbates interferences in organic solute rejection during bench-scale filtration

Materials in contact with purified (e.g., Drinking water, ultrapure-water) water are sources of dissolved organic carbon which can increase the regrowth potential. Reverse osmosis (RO) membrane materials are composed of flexible polymers that are also in contact with treated water, but their contribution to DOC and microbial regrowth in bench-scale filtration studies is not reported. Bench-scale filtration studies use less volumetric throughput compared to pilot and full-scale filtration systems, which likely exacerbates the influence of leachates on for DOC measurement in permeates. This limits our ability to determine if standard membrane preparation procedures ensure sufficient removal of leachable organics. Migration of DOC from 8 different reverse osmosis membrane filters was assessed by two different experimental approaches; (1) Immersion assay—membranes were soaked in a pasteurized, isotonic water solution with a low and constant concentration of DOC. (2) Volume throughput (VT) test—Permeate was collected at defined VT steps to determine the impact of VT on the washout of DOC during system start-up. Test water from the immersion assay and the filtration test were analyzed with respect to (i) DOC (mg/L), (ii) microbial regrowth potential (% increase in bacterial activity), (iii) DOC fractional composition via LC-OCD. The presented study identified variations in the leachability of DOC between commercial RO membranes. Leaching of DOC from RO membranes varied by supplier and membrane composition. Alfa Laval RO98 exhibited the highest release of DOC compared to the other membranes tested during the immersion assay, suggested by an increase in test water DOC from 0.3 mg/L to 7.43 ± 1.54 mg/L, (n = 3) within 48 h. Aquaporin biomimetic-based membranes had the lowest release of DOC after 48 h of immersion (0.702 ± 0.17 mg DOC/L, n = 3). Higher concentrations of leached DOC did not lead to a higher regrowth potential on the released solute. Results of the filtration test demonstrate that DOC leaching from the membrane can exceed the point of flux-stabilisation, which is the common endpoint of membrane preparation. In all membranes tested, the chemical composition of DOC was dominated by low molecular weight acids, indicative of monomers used during membrane interfacial polymerization. The implication of this study suggests that on bench-scale filtration less volumetric throughput compared to pilot-full scale filtration exacerbates the influence of leachates on for example DOC measurement in permeates; promoting interferences in DOC permeate measurement and corresponding rejection coefficient. Relevance for full-scale filtration systems is limited due to higher volume throughputs applied during system start-up and commissioning.

Development of quality control parameters for classical ayurvedic formulation: Vyoshadi Churna

Introduction: Ayurvedic formulations have a long history of use and have increased amazingly to follow the global interest in natural products. However, for many classical ayurvedic formulations, no quality parameters of standardization are available. Vyoshadi Churna, a classical Ayurvedic polyherbal formulation that cures indigestion and diarrhea, is being used since ancient times. However, there is no standard method available for its formulation and evaluation. Hence, the current study aims to develop the quality standards for Vyoshadi churna. Methods: The standard procedures for preparation and standardization of Churna described in Ayurvedic Pharmacopoeia of India (API) were used in the present study. Standardization includes pharmacognostical, chromatographic, and physico-chemical studies. The other essential studies were also done in accordance to API and World Health Organization guidelines to find the limits of microbial load, aflatoxin, heavy metals, and pesticide residue using modern analytical techniques such as gas chromatography-mass spectroscopy (MS) and inductively coupled plasma-MS/MS. Results: Vyoshadi Churna is of brown color and bitter in taste. Pharmacognostical studies have shown the characteristic features of almost each contributory crude drug. A unique chemical profile was also developed using toluene: ethyl acetate: methanol: formic acid (4:4:1:0.5) as the mobile phase to resolve maximum components. Furthermore, the standardized limits for the physicochemical studies, microbial load, heavy metals, aflatoxins, and pesticide residues were established and found in limits. Conclusion: This is for the first time when the standard operating procedure of Vyoshadi Churna was developed. Hence, the current technical work would be practically helpful for the future preparation and standardization study of Vyoshadi Churna.

Towards standardizing the preparation of test specimens made with material extrusion: Review of current techniques for tensile testing

Researchers and industries rely heavily on standardized testing methods to ensure products are designed and manufactured with high quality. With increasing use of additive manufacturing processes, such as material extrusion (MEX), there is a significant gap in material testing standards tailored for such components, and a lack of guidance on preparing appropriate test specimens with mesostructures that best represent the final part being analyzed. This paper aims to support the standardization of MEX material testing by reviewing the current methods used for preparing test specimens for tensile testing and proposing guidelines for implementation in a new standard. The need for standardization of MEX specimen preparation is addressed by analyzing the effects of slicing parameters on resulting tensile properties of the specimen. It is suggested that a standard should acknowledge these parameters, in addition to specimen geometry, toolpath optimization, printer and material specifications, so that they are appropriately selected for the test specimen by regarding the final part structure. Consideration of the proposed guidelines in a standardized method may enable comparisons between published results and support the development of MEX technology for use in advanced applications. • Material testing standards are incomplete for specimens made by material extrusion. • Test specimen preparation procedures found in literature are inconsistent. • Process parameters greatly influence tensile strength and stiffness. • Guidelines for a standard test specimen preparation procedure are proposed.

Restoring the surface layer of Bi2Sr2CaCu2O8+δ single crystals by current injection

In 2010, we reported a new method to electronically tailor the hole concentration of the high-Tc superconductor Bi2Sr2CaCu2O8+δ (Bi-2212). We discovered that current injection along the c-axis of Bi-2212 provides a convenient tool to reversibly increase the carrier concentration. In this study, we have investigated the current injection effect on the surface junction of Bi-2212 by a series of current injection experiments carried out in samples of slightly overdoped and of extremely underdoped Bi-2212 crystals. The c-axis transport characteristics of bulk and surface intrinsic Josephson junctions are examined in a wide range of doping by current injection. Before injection treatment, typically, our samples demonstrate non-superconducting characteristics across their surface junctions, because standard preparation procedures cause the topmost CuO2 layers to be heavily degraded by oxygen loss. The electronic doping enables to substantially restore the hole concentration of the topmost CuO2 layers. The contact resistance is significantly reduced, and the intrinsic junction at the surface gets back its superconducting tunneling characteristic. We examine systematically the doping results of surface and bulk junction stacks and discuss the enhancement of their superconducting properties.

Extraction of anabolic steroids with aqueous-organic mixtures from hexanic solutions

The extraction of anabolic steroids (1,4-androstadiene-3,17-dione; 17α-methyltestosterone; 19-norethiocholanolone; 4-hydroxytestosterone; 4-androstene-3,17-dione; 5α-androstane-3β,17β-diol; dehydroepiandrosterone; calusterone; clostebol; methandienone; methyldienolone; nandrolone; testosterone; epitestosterone; tibolone; 19-norandrostenedione; 1-methylene-5α-androstan-3α-ol-17-one) by aqueous-organic mixtures from hexane solutions was studied at the temperature (20 ± 1) °C. Based on the experimental data obtained, the partition ratios of anabolic steroids were calculated, which were used to optimise the standard sample preparation procedure in the process of determining the compounds under study in biologically active dietary supplements and specialised sports nutrition. It was found that the most effective and selective extractants from hydrocarbon solutions are water-acetonitrile mixtures containing from 10 to 20 % by volume of water. An extraction technique has been developed for sample preparation of biologically active dietary supplements for subsequent gas chromatographic determination of anabolic steroids in them using a triple quadrupole mass spectrometric detector. The proposed method is characterised by a standard deviation of 10–15 % and a detection limit about 10 μg/kg of biologically active dietary supplements, which makes it possible to reliably determine impurities of prohibited anabolic steroids in them.

Impact of Pre-Analytical and Analytical Variables Associated with Sample Preparation on Flow Cytometric Stainings Obtained with EuroFlow Panels.

Simple SummaryObjective interpretation of flow cytometry may be hampered by a lack of standardized sample preparation procedures. The EuroFlow consortium conducted a series of experiments to determine the potential impact of different pre-analytical and analytical factors on the variability of results in terms of relative cell populations distribution and marker expression levels. The experiments were performed on healthy donors and patients with different hematological malignancies (e.g., acute leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome) to mimic real-world clinical settings. Overall, the results showed that sample storage conditions, anticoagulant use, and sample processing protocol might need to be tailored for sample and cell type(s), as well as to the specific markers evaluated. However, defining of well-balanced boundaries for storage time to 24 h, staining-acquisition delay to 3 h, and choosing a washing buffer of pH within the range of 7.2 to 7.8 would be a valid recommendation for most applications and circumstances described herein.Objective interpretation of FC results may still be hampered by limited technical standardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on different cell populations, from both healthy donors and patients’ samples with distinct hematological malignancies. The use of different anticoagulants; the time interval between sample collection, preparation, and acquisition; pH of washing buffers; and the use of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) staining protocols, were evaluated. Our results showed that only monocytes were represented at higher percentages in EDTA- vs. heparin-anticoagulated samples. Application of SM or SM+CY protocols resulted in slight differences in the percentage of neutrophils and debris determined only with particular antibody combinations. In turn, storage of samples for 24 h at RT was associated with greater percentage of debris and cell doublets when the plasma cell disorder panel was used. Furthermore, 24 h storage of stained cells at RT was selectively detrimental for MFI levels of CD19 and CD45 on mature B- and T-cells (but not on leukemic blasts, clonal B- and plasma cells, neutrophils, and NK cells). The obtained results showed that the variables evaluated might need to be tailored for sample and cell type(s) as well as to the specific markers compared; however, defining of well-balanced boundaries for storage time, staining-to-acquisition delay, and pH of washing buffer would be a valid recommendation for most applications and circumstances described herein.

PROCESS VALIDATION AND GC ANALYSIS OF PATHADISURA, A FERMENTED AYURVEDIC PRODUCT USED IN SHWAS VYADHI [BRONCHIAL ASTHMA

Pathadisura [PS] is an ayurvedic fermented formulation, prescribed for respiratory disorders. It contains Suramanda prepared from (Rice grains), Guda (Jaggery) and herbal drugs viz Patha Murva, Rasna, Saral, Devdaru. The scientific preparation method and quality parameters of PS are not documented yet. Thus present study is aimed to develop and validate the preparation method and to generate quality standards for PS. Preparation of Sura, Suramanda and PS was done as per standard protocols of ayurvedic pharmaceutics blended with modern techniques. All herbal drugs, Sura and PS were tested with standard analytical methods. Obtained values for herbal drugs were within limits as per API standards, signify purity of plant materials. Study result provides organoleptic, physico-chemical values and gas chromatography quality standards for Sura and Pathadisura. Standard operating procedure for preparation of Sura and Pathadisura is developed and validated by using standard methods of analysis. It could be used in further studies. KEYWORDS: Pathadi Sura, Sura, Annamanda, Fermentation, GC Analysis.

Effectiveness of preoperative bath using chloroxylenol antiseptic soap on the incidence of post emergency cesarean section surgical site infection at Mbarara Regional Referral hospital, Uganda: a randomized controlled trial.

IntroductionSurgical Site Infections (SSIs) constitute 15%-45% of hospital acquired infections in sub-Saharan Africa. Cesarean section (CS) increases the risk of developing sepsis by 5-20 times and is highest when the operation is emergency. Therefore, the purpose of this study was to measure the effect of chloroxylenol in reducing the incidence of post cesarean SSIs at Mbarara Regional Referral Hospital (MRRH).Methodsa randomized controlled trial was conducted at MRRH maternity ward and mothers were randomized into either control or intervention arms. The intervention was a complete body bath with chloroxylenol antiseptic soap before the operation, while the control arm participants received a standard ward pre-operative preparation procedures. All participants were followed up for 30 days and assessed using an SSI screening tool.Resultsninety-six women were randomized, and 48 were assigned to each arm. The overall incidence of SSI was 30.21%. The incidence of SSI was significantly lower in the intervention compared to the control arm (6.25% in the intervention arm versus 54.17% in the control arm) (p-value <0.001). Chloroxylenol bath was protective of SSI with a 90% risk reduction for SSI (95% confidence interval of 67%–97%).Conclusiona preoperative bath with chloroxylenol for pregnant mothers is associated with a significantly lower risk of post cesarean section surgical site infections. Health facilities with a high burden of post SSI should consider adding this simple and effective intervention to the existing infection prevention measures. Clinical Trials.gov registration (NCT03544710).

Clinical Evaluation of Plasma Separation Cards as a Tool to Collect, Store, and Test Blood Samples for Hepatitis B and C Serological Markers.

The plasma separation card (PSC) is a new device for collecting finger-pricking-derived small amount of blood in a solid support that is stable at room temperature and can be archived, mailed, and processed at a later time. This tool can facilitate screening at risk populations located in rural areas without local health care infrastructures. We evaluated the performance of PSC in the collection and preparation of blood samples for the determination of hepatitis B and C serological markers. Blood obtained from 334 consecutive patients referred for the detection of hepatitis B surface antigens (HBsAg), hepatitis B surface antibodies (anti-HBs) and hepatitis C antibodies (anti-HCV) was analyzed in parallel using standard (STD) and PSC-based sample collection and preparation procedures. Results obtained from STD or PSC processed samples were compared for their detection rate and correlation. Using STD, we detected 5 samples positive for HBsAg, 150 for anti-HBs, and 23 for anti-HCV with a rate of concordance with PSC of 100%, 100%, and 91% respectively. The 100% concordance observed for anti-HBs was based on a cutoff of 2.6 IU/L for PSC-derived sample corresponding to the 10 IU/L threshold associated with immunity to hepatitis B. STD and PSC showed a good correlation (R2 = 0.85) in the detection of anti-HBs titers. The 2 anti-HCV PSC negative samples had no detectable viremia. These data confirm the utility of PSC as a tool to support viral hepatitis screening programs in rural areas lacking local clinical infrastructures and testing facilities.

Virtual Reality Tour to Reduce Perioperative Anxiety in an Operating Setting Before Anesthesia: Randomized Clinical Trial.

BackgroundPerioperative anxiety is a major burden to patients undergoing surgeries with general anesthesia.ObjectiveThis study investigated whether a virtual operating room tour (VORT) before surgery can be used to ameliorate perioperative anxiety.MethodsWe employed a randomized parallel-group design with 2 study arms to compare VORT to the standard operation preparation procedure. The study included 84 patients. A validated inventory (state-trait operation anxiety-state) was used to assess perioperative state anxiety before (T1) and after (T2) surgery. In addition, trait operation anxiety was evaluated with an additional validated inventory (state-trait operation anxiety-trait). Moreover, user ratings on the usefulness of VORT were assessed with an evaluation questionnaire. Study arms were compared for perioperative state anxiety with two-tailed independent samples t tests. Subjective ratings were correlated with STOA-Trait values to investigate possible associations between perioperative anxiety with perceived usefulness.ResultsThere were no significant differences in perioperative state anxiety between VORT and standard operation preparation procedures before and after the surgery. Nonetheless, patients’ ratings of VORT overall were positive. The tour was perceived as useful and, therefore, showed acceptance for VR use. These ratings were unrelated to the degree of perioperative anxiety.ConclusionsThe subjective benefit of VORT could not be explained by a reduction of perioperative anxiety. Instead, VORT appears to serve the need for information and reduce uncertainty. In addition, VORT is perceived as beneficial regardless of the age of the patients. Considering this effect and the manageable organizational and financial effort toward implementation, the general use of VORT can be recommended.Trial RegistrationClinicalTrials.gov NCT04579354; https://clinicaltrials.gov/ct2/show/NCT04579354

Genome-wide identification of 5-methylcytosine sites in bacterial genomes by high-throughput sequencing of MspJI restriction fragments.

Single-molecule Real-Time (SMRT) sequencing can easily identify sites of N6-methyladenine and N4-methylcytosine within DNA sequences, but similar identification of 5-methylcytosine sites is not as straightforward. In prokaryotic DNA, methylation typically occurs within specific sequence contexts, or motifs, that are a property of the methyltransferases that “write” these epigenetic marks. We present here a straightforward, cost-effective alternative to both SMRT and bisulfite sequencing for the determination of prokaryotic 5-methylcytosine methylation motifs. The method, called MFRE-Seq, relies on excision and isolation of fully methylated fragments of predictable size using MspJI-Family Restriction Enzymes (MFREs), which depend on the presence of 5-methylcytosine for cleavage. We demonstrate that MFRE-Seq is compatible with both Illumina and Ion Torrent sequencing platforms and requires only a digestion step and simple column purification of size-selected digest fragments prior to standard library preparation procedures. We applied MFRE-Seq to numerous bacterial and archaeal genomic DNA preparations and successfully confirmed known motifs and identified novel ones. This method should be a useful complement to existing methodologies for studying prokaryotic methylomes and characterizing the contributing methyltransferases.